AU7971294A - (plasmodium falciparum) ribonucleotide reductase, encoding dna, and inhibitors - Google Patents

Description

"PLASMODIUM FALCIPARUM RIBONUCLEOTIDE REDUCTASE, ENCODING DNA, AND INHIBITORS"

Field of the Invention This invention relates to the ribonucleotide reduc¬ tase of Plasmodium falciparum (Pf RR) , to the subunits of Pf RR (Pf Rl and Pf R2) , and to compounds comprising peptides derived from the Pf R2 C-terminus sequence that inhibit the action of protozoal RR.

Background of the Invention Antimalarial Therapy

Malaria is a leading cause of morbidity and mortality worldwide. It accounts for more than one million deaths annually. Malaria caused by the parasite Plasmodium falciparum (hereinafter "Pf"), is the most deadly of four malarial parasites.

The Anopheles mosquito-borne Pf malaria has frustrated scientists formore than a century in the worldwide quest to control malaria. The problem is compounded by emergence of drug resistant strains and slow progress in developing an effective vaccine. Thus, more effective antimalarial agents are needed.

According to the WorldHealthOrganizationmalaria is a major disease, that is steadily spreading today. The WHO estimates that 270 million people now carry the parasite. In 103 countries people are currently at risk from malaria. This amounts to nearly half of the world's population.

Ribonucleotide Reductase

Ribonucleotide reductase (hereinafter "RR"), is a ubiquitous biological catalyst necessary for DNA biosyn¬ thesis in all forms of life. It controls an early rate- limiting reaction in de novo DNA synthesis. This single enzyme catalyses the first step in each of the parallel pathways to the four deoxyribonucleoside triphosphates. By reducing ribonucleotides to their corresponding deoxy- ribonucleotides, RR provides cells with precursors needed for DNA biosynthesis and repli-cation. Hence, viral, bacterial and eukaryotic (e.g., protozoan and mammalian) cellular proliferation inter alia is dependent upon the presence of the active enzyme. Another requirement for activity is a pool of ribonucleotide diphosphates (NDPs) and triphosphates (NTPs) .

RR occurs in two subunits, α and β, which must be associated with each one for activity. Subunit is comprised of two identical Rl subunits. Subunit β is comprised of two identical R2 subunits. The Rl and R2 subunits generally have molecular weights of 84-90 and 37-45 kDa, respectively. Each subunit plays a critical role in the reduction process of DNA biosynthesis, and each is coded for by a distinct gene. Separation of subunit types leads to complete loss of reduction activity, which can be recovered only upon reassociation of the holoenzyme.

The fraction of fully conserved amino acid residues among all known RR sequences is low (6% in Rl and 5% in R2) . Eukaryotic R2 sequences from unrelated organisms may be related for up to seven residues from the C-terminus, but the sequences are usually not con-served after the eighth amino acid residue from the C-terminus. Therefore, peptide RR inhibitors are thought to be species specific, or at least specific for related species. (Yang et al . , FEBS Lett. 272. 61-64 (1990) ) . However, R2 sequence identity may occasionally be quite high between related organisms. Homology between mouse and human forms exceeds 90%. (Pavloff et al. , J. of DNA Sequencing and Mapping 2, 227-234 (1992)) . For example, it has been demonstrated that the acylated peptide Ac- (SEQ ID NO:6) , corresponding to the C-terminus of mouse R2, inhibits human RR with an IC₅₀ of about 10-20 μM. It is anticipated that an acylated heptapeptide cor- esponding to the C-terminus of human R2 would inhibit human RR with a similar IC._n. Ordinarily, a short synthetic peptide having a sequence the same as "the R2 C-terminus of a species should inhibit that species RR with an IC₅₀ of 10-20 μM. For example, an acetylated synthetic peptide of nine amino acid residues, identical to the herpes simplex R2 C-terminus sequence, was used to inhibit viral RR activity by competing with herpes simplex R2 for association with herpes simplex Rl. The nonapeptide, Ac- (SEQ ID NO:5) had an IC₅₀ of 10-20 μM (Dutia et al. , Nature 321, 439-441 (1986) ; Cohen et al. , Nature 321, 441-443 (1986); Clements et al. , Virology 162. 270-273 (1988); and Paradis et al. , J. Biol. Chem. 263, 16045-16050 (1988) ) .

The Pf parasite develops in erythrocytes. Mature human erythrocytes do not have RR to produce DNA, therefore, parasitic RR function will not be replaced if the parasitic RR is deactivated. Deactivation of parasitic RR without deactivating the host enzyme in the host's non-erythrocytic cells is a feasable and particularly attractive therapeutic goal.

Pf RR has not been isolated or characterized; nor have its respective genes been isolated or sequenced. Isolation is expected to be difficult since protozoan RR activity is present in host cells only at very low levels.

Summary of the Invention

The invention provides a DNA segment comprising a sequence according to SEQ ID NO:l or SEQ ID NO:3, wherein the DNA segment consists essentially of a nucleotide sequence encoding for a polypeptide according to SEQ ID NO:2 or SEQ ID NO:4, respectively. SEQ ID NO:2 and SEQ ID NO:4 comprise the Pf Rl and Pf R2 subunits of Pf RR, respectively.

In another embodiment, the present invention provides a plasmid transfer or storage vector comprising a DNA segment consisting essentially of a nucleotide sequence encoding for a polypeptide having essentially the sequence according to amino acid SEQ ID NO:2 or SEQ ID NO:4. Such a plasmid transfer or storage vector may comprise a DNA segment having essentially the nucleotide sequence according to SEQ ID NO:l or SEQ ID NO:3. In another embodiment, the present invention provides a host cell transformed by a vector comprising a DNA segment consisting essentially of a nucleotide sequence according to SEQ ID NO:l or SEQ ID NO:3. Under culture conditions the host cell is capable of producing a polypeptide having essentially the amino acid sequence according to SEQ ID NO:2 or SEQ ID NO:4.

In a further embodiment the present invention provides a method for producing a polypeptide having essentially a sequence according to SEQ ID NO:2 or SEQ ID NO:4, such method comprising the steps of: cloning into a host cell a DNA segment encoding for a polypeptide having essentially the amino acid sequence according to SEQ ID NO:2 or SEQ ID NO:4, thereby producing a transformed host cell; culturing the transformed host cell under such conditions as to produce the polypeptide; and isolating or purifying the polypeptide from the culture media and/or the transformed host cells.

In another embodiment, the present invention provides antimalarial compounds which are specific inhibitors of P. falciparum ribonucleotide reductase and which have little or no toxicity toward humans. The antima-larial compounds comprise peptides having a chain length of from seven to about 400 amino acids, preferably from seven to about 100 amino acids, and most preferably, from seven to about 20 amino acids. These compounds have the following formula:

Y-X₁-Phe-X₃-Leu-X₄-Phe-X₂; (I)

wherein Y is H or a blocking group on the peptide N-terminal amino group; X is from zero to about 393 amino acids, provid¬ ed when X-_L is more than one amino acid, the amino acids are either the same or different; X₂ is OH or at least one amino acid, provided when X₂ is more than one amino acid, the amino acids are either the same of different; X₃ is any amino acid; X₄ is from zero to three amino acids, provided when X₄ is more than one amino acid, the amino acids are either the same or different; and further providing that the total amino acids of the sequence corresponding to the segment -Leu-X₄-Phe-X₂ of formula (I) is five amino acids.

Preferred compounds according to formula (I) are those wherein X_x is from zero to about 93 amino acids. More preferred compounds are those compounds wherein X_x is from zero to about thirteen amino acids, and Y is a blocking group on the peptide N-terminal amino group when X₁ is from zero to about three amino acids.

Particularly preferred compounds according to formula (I) are those compounds wherein X_lf X₂, and X₃ are defined as above, and X₄ is a group selected from the group consisting of one of the following sequences:

Xaa-Xaa-Xaa; (II)

Xaa-Xaa-Glu; (III)

Xaa-Thr-Xaa; (IV)

Asn-Xaa-Xaa; (V)

Xaa-Thr-Glu; (VI)

Asn-Xaa-Glu; (VII)

Asn-Thr-Xaa; (VIII)

and

Asn-Thr-Glu (IX) ;

wherein each Xaa of sequences II-VIII is the same or different and is independently any amino acid. Most preferred compounds are such compounds wherein X₃ is Cys. Even more preferred are compounds which comprise a sequence according to SEQ ID NO:7.

Preferred antimalarial compounds according to the present include, but are not limited to, blocked peptides derived from the Pf R2 C-terminus (SEQ ID NO:2) . In another embodiment the present invention provides unblocked interme¬ diate peptide compounds wherein X_x is from zero to about three amino acids, which are useful for preparing blocked deriva- tives.

In a yet further embodiment, antimalarial com¬ positions are provided which comprise a pharmaceutically acceptable carrier and at least one compound according to the invention having a peptide length from 7 to about 20 amino acids, which inhibits Pf RR reduction of ribonucleotides to 2' -deoxyribonucleotides.

In another embodiment, the present invention provides a method of therapeutic treatment and prevention of malaria caused by P. falciparum by controlling the prolifer- ation of P. falciparum comprising administering to a patient an effective amount of at least one antimalarial compound according to the invention having a peptide length from 7 to about 20 amino acids, such that Pf RR reduction of ribonucleo¬ tides to 2' -deoxyribonucleotides is inhibited. In another embodiment the present invention provides a recombinant Pf RR enzyme comprising Pf Rl, or a functionally equivalent polypeptide thereof and/or Pf R2, or a functional equivalent thereof. Preferably said recombinant Pf RR enzyme has the formula (Pf R2)₂-(Pf Rl)₂ wherein said Pf R2 and Pf Rl each comprise a sequence according to SEQ ID NO:2 and SEQ ID NO:4, respectively.

In a further embodiment the present invention provides amethod for screeningpotential antimalarial agents for antimalarial activity which comprises contacting said potential antimalarial agents with Pf RR and assaying for inhibition of Pf RR enzymatic activity.

In another embodiment the present invention provides a method for screeningpotential antimalarial agents for antimalarial activity which comprises contacting said potential antimalarial agents with Pf Rl and assaying for binding of said agents to Pf Rl, optionally in the presence of Pf R2. In a preferred method said potential antimalarial agent is contacted with Pf Rl in the presence of Pf R2, or is contacted with Pf Rl followed by contact with Pf R2, and assayed for the blocking of Pf Rl binding to Pf R2.

The present invention further provides a method of diagnosing the presence or absence of a malarial parasitic infection in a host which comprises: a) obtaining a blood sample from a host in need of diagnosis for the presence or absence of a malarial parasite; b) contacting said sample with a PCR primer pair which is capable of amplifying a Rl or R2 target DNA segment of said malarial parasite, which target DNA segment is unique as compared to said host DNA, under conditions such that said primer pair will amplify said target DNA segment if said target DNA segment is present; c) contacting said sample with a polynucleo-tide probe which is capable of specifically hybridizing with said target DNA segment, under conditions such that said polynucle- otide probe will hybridize with said target DNA segment if said target DNA segment is present; and d) assaying said sample to determine the presence or absence of said malarial parasite.

Description of the Figures

Figure 1 is a graph showing the inhibition of calf thymus/mouse ribonucleotide reductase activity by the acylated polypeptide, Ac- (SEQ ID NO:7) .

Detailed Description of the Invention Unless specifically stated otherwise, all sequences are indicated as follows. All peptide sequences have their N-terminus to the left and their C-terminus to the right. All nucleotide sequences have their 5' -terminus to the left and their 3'-terminus to the right. Further, when a single dash is shown in any polypeptide sequence it indicates a single peptide covalent bond or a mimic bond bridging the two amino acid residues. The compounds according to formula I include both polypeptides comprised of ordinary amino acids and amino acid derivatives which are used to produce conformational mimics of the active polypeptide structure. The term "amino blocking group" as referred to above, or in the text and claims below, means any group capable of blocking an N-terminal amino group of a polypeptide, by replacing a hydrogen atom. For example, the blocking group may comprise an acetyl group.

Based upon the isolation of Pf R2 genomic DNA and elucidation of the corresponding amino acid sequence, compounds have been derived which are protozoan-specific. These compounds do not significantly inhibit mammalian RR enzyme. Thus, they are useful for controlling proliferation of protozoan parasites in mammals. The peptides are specific inhibitors of Pf RR and are believed to have little or no toxicity toward humans. Inhibition of Pf RR halts the malarial parasite's intraerythrocytic maturation since mature erythrocytes do not have their own RR.

The N-terminally blocked antimalarial peptides may be prepared from peptide intermediates derived from the Pf R2 C-terminus and from functionally equivalent derivative peptides. When X is from zero to about three amino acids, the N-termini of the intermediate peptides are preferably blocked to provide better mimics of the intact native Pf R2 protein. Blocking the N-terminal amino group can lead to im¬ proved Pf Rl binding and inhibition of RR activity. Any blocking group which can be attached to an amino group either directly or through an intermediate linking group maybe used. Acyl groups are preferred, of which acetyl or benzoyl groups are most preferred. Acetylating and benzoylating agents, and procedures for blocking amino terminal groups of peptides, are well known to those skilled in the art.

Isolation and Characterization of Pf RR DNA

We have found that the genes encoding Pf Rl and Pf R2 are both located on chromosome 14 of the Plasmodium falciparum eukaryotic protozoan. By contrast, in the mouse and human eukaryotic RR, the subunit genes are located on different chromosomes (Brissenden et al. , U. Exp. Cell. Res. 174, 302-308, (1988); Yang-Feng et al. , U. Genomics 1, 77-86 (1987) ) . Northern analysis of messenger RNA derived from synchronized cultures of Pf shows a single transcript of approximately 2100 and 5200 nucleotides per R2 and Rl respectively. The R2 transcript appears approximately 12 hours earlier, and persists approximately 12 hours longer, than the Rl transcript. This pattern of transcript accumula¬ tion is different from that seen in the mammalian (mouse) sys¬ tem, where levels of both Rl and R2 transcripts rise and de¬ cline in parallel as cells progress through M-phase into G₂+M phase (Bjorklund et al. , Biochemistry 29, 5452-5458 (1990)) . Characterization of the R2 DNA sequence and deri¬ vation of the corresponding polypeptide sequence was carried out as follows.

Parasites were obtained and stored as described by Taraschi, et al. Science 232 102-104 (1986) . Genomic DNA was purified essentially as described by Medrano, et al.. Biotechnigues 8., 43-45 (1990) with minor modifications to optimize yields.

Infected RBCs were collected by centrifugation, rinsed and then incubated in a lysing solution to lyse the RBCs and release the parasites. Freed parasites were rinsed and incubated with RNase and a protease to digest RNA and pro¬ teins present in the mixture. After proteins and RNA were removed, the DNA was purified and amplified with polymerase chain reaction (PCR) primers designed to amplify R2 DNA. After amplification, the resulting DNA was made blunt-ended with a T4 polymerase, kinased with T4 polynu- cleotide kinase and ligated to a plasmid. The recombinant plasmids were isolated by standard techniques.

The Pf R2 N-terminal nucleotide sequence was iso- lated from the recombinant plasmid genomic Pf DNA by anchored PCR (APCR) . The genomic Pf DNA was digested with Xbal. Frag¬ ments were selected and gel purified from agarose gel. Size- selected DNA was ligated to a plasmid and samples of the liga¬ tionmixwere amplifiedwith PCRprimers. Resulting fragments were gel purified, reamplified and directly sequenced by chain termination reactions via procedures set forth in Thein, Comments (USB Technical Guide) JL6_. 8 (1989) . The Pf R2 C-terminal nucleotide sequence was iso^¬ lated from the recombinant plasmid genomic DNA by forced cloning and screening of colonies as follows.

Genomic DNA was digested with Asel and Xbal enzymes and fragments were size-selected from agarose gel. A Ndel site was inserted the EcoRl site of Blue-script KS+ via Ndel- EcoRl linkers. The digested genomic Pf DNA was ligated into KS+ digested with Xbal and Ndel (digested KS+ is designated Bluescript KS-Ndel) . DH5α E. coli cells were transformed by inserting the ligation mix. Colonies were screened for positive clones by standard procedures with a fragment of R2 amplified from high molecular weight DNA with PCR primers.

The positive clones were isolated and lysed to release the plasmids, which were then digested to yield Pf DNA fragments, the resulting DNA fragments were sequenced as described above for the N-terminal segment.

The entire Pf R2 genomic DNA sequence is set forth in SEQ ID NO:l, and the amino acid sequence for Pf R2 is set forth in SEQ ID NO:2. There is no evidence of introns in Pf R2. By comparison, mouse R2 contains 10 exons and 9 introns (Thelander et al. , Embo J. 8, 2475-2479 (1989)) .

The DNA sequence for Pf Rl was obtained substan¬ tially in the manner described above for obtaining the Pf R2 DNA sequence. The polypeptide sequence coded for by the Pf Rl DNA sequence, was then readily obtained by conventional procedures. The entire Pf Rl genomic DNA sequence is set forth in SEQ ID NO:3, and the amino acid sequence for Pf Rl is set forth in SEQ ID N0:4. In SEQ ID NO:3, nucleotides 1-63 are a non-coding 5' end; nucleotides 64-2193 code for amino acids corresponding to SEQ ID NO:4 positions 1-710; nuc¬ leotides 2194-2357 are a non-coding intron; nucleotides 2358- 2660 code for amino acids corresponding to SEQ ID NO:4 posi¬ tions 711-810; and nucleotides 2661-2663 are a non-coding terminating 3' end. SEQ ID NO:40 is a DNA segment coding for a polypeptide having a sequence according to SEQ ID NO:4 and corresponds to SEQ ID NO:3, with the non-coding nucleotide seg-ments removed. The DNA segment and polypeptide according to SEQ ID NO:3 and SEQ ID N0:4, respectively, are useful as intermediates to form the recombinant Pf RR complex and as reagents to develop and test antimalarial agents.

Recombinant Production of the Pf Rl Subunit The Pf DNA segment that codes for the Pf Rl subunit, may be cloned in a baculovirus system, essentially as described by Salem, et al. , FEBS Lett.. 323, No. 1,2, 93-95 (1993) , as follows. A. Obtaining and amplifying a DNA segment coding for the Pf Rl subunit

The Pf Rl DNA segment, having a sequence according to SEQ ID N0:3, is first obtained from parasites that are maintained and stored as describedby Taraschi, et al. Science 232 102-104 (1986) . Genomic DNA is purified essentially as described by Medrano, et al. , Biotechnigues 8., 43-45 (1990) with the following modifications. Infected red blood cells

(RBCs) from culture plates are collected by centrifugation and rinsed with phosphate buffered saline (PBS) . The packed, infected RBCs are resuspended in PBS. Saponin is added followed by incubating to release the parasites.

Freed parasites are rinsed with PBS, resuspended in lysis buffer and incubated at 37°C. RNase and protease are added to the solution which is incubated to digest the RNA and protein present. The digested proteins are pre¬ cipitated by adding salt to the solution. The solution is then extracted with solvent such as phenol/chloroform. DNA is precipitated with ethanol, washed and resuspended in water.

Two sets of polymerase chain reaction (PCR) primer pairs are used to amplify Pf Rl exons. The first pair is complementary to the N-terminus of SEQ ID NO:3 and a sequence adjacent to the 5' end of the intron. The second pair is complementary to a sequence adjacent to the 3' end of the intron and to the C-terminus of SEQ ID NO:3. The primer pairs are designed to amplify Pf Rl DNA exons and to add Nhel re¬ striction sites to the N-terminus and C-term-inus of the Pf Rl DNA segments. Samples of total genomic DNA, obtained from Pf as described above, are amplified via PCR with the PCR primers. Ligation of the two exons is performed by standard methods. B. Construction of the pBlueBac2-Pf Rl vector The amplification product from the above ampli¬ fication process is digested with Nhel and inserted into the Nhel site of pBlueBac2 (InVitrogen, San Diego, CA) to produce a Pf Rl vector (pBlueBac2-Pf Rl) . Spodoptera frugiperda (Sf9) cells (InVitrogen, San Diego, CA) are transformed with the recombinant plasmid using the cationic liposome method (Hartig et al.. Biotech.. 11, 310-313 (1991) . Positive clones are selected by beta-galactosidase blue/white screening and subsequently grown in culture dishes. Plaques are subjected to PCR analysis with primers complementary to the polyhedron loci to select those free of non-recombinant baculovirus.

C. Growth and maintenance of Sf9 cells Sf9 cells are cultured in Excell 400 medium (JRH

Scientific, Lenexa, KS) supplemented with heat inactivated fetal calf serum (FCS) (Bethesda Research Labs, Gaithersburg, MD) gentamycin and Fungizone that has been sterile filtered. Cells grown in spinner flasks are also supplemented with 0.1% Pluronic F-68 (JHR Scientific, Lenexa, KS) to reduce shear damage.

D. Expression of recombinant Pf Rl

Original stocks of recombinant virus, produced as above, are used to infect virgin Sf9 cells to screen for protein production and generate the second generation of virus. Aliquots of the infected Sf9 cells are removed and assayed intermittently for expression of recombinant Pf Rl protein. Plaques producing Pf Rl protein without viral contamination of the protein are selected for further study. Larger culture volumes are grown and harvested by centrifugation to produce a cell pellet.

E. Purification of Recombinant Pf Rl The cell pellet collected from the culture is resuspended in lysis buffer and subjected to cycles of freeze- thaw. The lysate is centrifuged to produce a supernatant which is loaded directly onto a Sepharose affinity column (to which a short peptide corresponding to Pf R2 C-terminus is bound) using the method described in Yang, et al■ , FEBS Lett. 272 61-64 (1990)) . The column is washed with a buffer and then with a small volume of buffer + KC1 to elute the recombinant Pf Rl. The column is regenerated by treatment with 6 M guanidine-HCl. Column fractions are monitored by the Bradford assay (Bradford, Id. (1976)) .

F. Recombinant Pf Rl Identification

A partial N-terminal sequence of the purified protein is determined by the Edman method (Edman, et al. Eur. J. Biochem. 180-91 (1967) using an Applied Biosystems model 473A sequencer. SDS polyacrylamide gel electrophoresis (SDS- PAGE) is performed using 7.5% acrylamide. Western blots are performed as described in Rubin et al. J. Biol. Chem. 265, 1199-1207 (1990) using a polyclonal antibody raised against a synthetic protein segment of Pf Rl which is unique to Pf Rl as obtained from SEQ ID NO:3. Protein concentration is determined according to the Bradford assay (Bradford, Anal. Biochem. 72 248-254 (1976) .

Recombinant Production of the Pf R2 Subunit

Expression of recombinant Pf R2 is based on transcription of T7 RNA polymerase according to the method of Mann, et al.. Biochem. 30 1939-1947 (1991)) . The Pf R2 protein is expressed and purified to homogeneity following a rapid and simple purification procedure. The Pf R2 protein is expressed from T7 RNA polymerase responsive plasmids, which are constructed by using standard molecular cloning tech¬ niques. Plasmids are propagated in E. coli strains grown in LB medium at 37°C, in the presence of carbenicillin. Transfections are performed by using the procedure according to Hanahan (J. Mol. Biol. 166 557-580 (1983)) .

A. Obtaining and Amplifying a DNA Segment Coding for the Pf R2 Subunit

The Pf R2 DNA segment, having a sequence according to SEQ ID NO:l, and coding for Pf R2, is first obtained from parasites in the same manner as the Pf Rl segment is obtained above. A polymerase chain reaction (PCR) primer pair terminated with a Nhel restriction site, for example, and complementary to the N-terminus and C-terminus of SEQ ID NO:l, respectively, is designed. The primers amplify Pf R2 DNA and add a Nhel restriction site to the N-terminus and to the C- terminus of the Pf R2 DNA segments. The Pf R2 DNA segment is amplified by PCR in the same manner as for Pf Rl, above.

B. Construction of the pET3a-Pf R2 Vector

The Pf R2 PCR amplification product is cleaved with Nhel restriction enzyme to produce a Nhel Pf R2 DNA fragment construct. This Pf R2 construct is transferred into the T7 expression vector pET3a (Rosenberg et al. , Gene 56 125-135 (1987) ; Studier et al . , Methods Enzvmol.. 185 60-89 (1990)) opened with the same enzyme. The resulting pET3a-Pf R2 vector is transfected into an E. coli strain which contains a lac- (IPTG) -inducible, chromosomal copy of the T7 RNA polymerase gene. The plasmid pET3a and the bacterial strain BL21(DE3) , are described in Studier, et al .. Methods Enzvmol . , 18560-89 (1990) .

C. Growth and Maintenance of E. coli cells Typically, BL21 (DE3) E. coli bacteria transfected with the pET3a-Pf R2 plasmid are logarithmically grown in LB medium containing carbenicillin. The LB medium is infected with a small quantity of overnight cultures of BL21(DES) bacteria, which is then cultured under agitation at 37°C. The LB medium containing the BL21(DES) bacterial culture is supplemented with IPTG when the absorption density is at A₅₉₀ is 0.6-1.0. The BL2KDE3) bacteria may then be cultured for a few additional hours before harvesting.

D. Expression of Recombinant Pf R2 Protein Pf R2 protein is produced by induction of log¬ arithmically growing BL21(DE3) bacteria containing plasmid pET3a-Pf R2 as described above. After incubation as described above, the cultures are chilled and centrifuged to produce a pellet. The pellet from the first centrifugation is gently resuspended in a buffer and again centrifuged. The pellet from the second centrifugation is frozen in liquid nitrogen and stored frozen until recombinant Pf R2 is to be purified from the pellet.

E. Purification of Recombinant Pf R2 Protein Each purification protocol involves disintegration of frozen bacteria containing the Pf R2 protein, extraction into a buffer, precipitation of nucleic acids with streptomy- cin sulfate, ammonium sulfate precipitation, and anion- exchange chromatography. All operations are performed in a cold room.

Pf R2 preparation, which is a modification of the previously published procedure for preparing recombinant I _ coli R2 (S δberg et al.. Biochem. 2615658-5662 (1986)), is described in detail where it differs from the published method.

Protein concentrations and purity may be assessed by Coomassie Brilliant Blue dye binding, with reference to a bovine serum albumin standard (Bradford, et al. , Anal. Biochem.. 72 148-254 (1976)), combined with laser densito¬ metric (LKB Pharmacia, Piscataway, NJ) scanning of TCA- precipitated samples separated on 10% SDS-polyacrylamide gels (Engstrδm et al. , Biochem.. 18 2941-2948 (1979)) . Concentrations of highly purified proteins are measured by light absorbance and calculated from extinction coefficients, as discussed below.

Frozen pellets of E. coli bacteria containing plasmid pET3a-Pf R2 are obtained from the above cultures and centrifugation. The pellets are finely ground in a mortar with cold aluminum oxide (Sigma Chemical Co. , St. Louis, MO) , using liquid nitrogen as necessary to prevent thawing. The powder is either stored frozen or directly mixed and thawed rapidly on ice in extraction buffer and centrifuged. Strepto- mycin sulfate is added to the supernatant to a final concentration of 2.5% while stirring on ice, followed by centrifugation. Solid ammonium sulfate is then added to the supernatant and the precipitate is recovered by centrifugation. After the precipitate is dissolved in extraction buffer, the extract is iron-reactivated (see below) or directly equilibrated (apo-Pf R2 preparations) in a buffer on a column containing Sephadex G-25 medium. Then about 30 mg of partially purified Pf R2 is loaded onto a DEAE-cellulose column (DE 52, Whatman Laboratory Products, Inc., Clifton, NJ) , previously equilibrated with 10 mM potassium phosphate, pH 7.0, 30 mM KCl, and 1 mM EDTA. The column is washed with the same buffer and Pf R2 is then eluted in 3-4 column volume of 10 mM potassium phosphate, pH 7.0, 70 mM KCl, and 1 mM EDTA. The protein eluate is frozen directly or is recovered by overnight dialysis at a low temperature against saturated ammonium sulfate. The eluted or dialysizedprotein is centri¬ fuged, dissolved in TrisHCl, pH 7.6, and is then stored frozen in aliquots.

Enzyme Activity of Recombinant Pf Rl and R2 Preparations

A. Recombinant Pf RR Activity Assay Ribonucleotide reductase activity is assayed at 37°C using the method of Moore and Peterson, Biochem. 132904- 2907 (1974) with minor modifications. Briefly, assay mixtures contain 60 mM HEPES, pH 7.6, 26 mM DTT, 7 mM NaF, 5 mM Mg(OAc)₂, 3 mM ATP, 0.05 mM FeCl₃, and either 10 μg of recombinant Pf R2, which is prepared using the published R2 recombinant method of Mann et al. Biochem. 30 1939-1947 (1991) , or an equivalent volume of a buffer. For CDP reductase assays, 0.05 mM [5-³H] -CDP (20 Ci/mol) is added to the assay mixture, while for GDP reductase assays 1.5 mM dTTP and 0.02 mM [2,8-³H] -GDP (45Ci/mol) is added to a final volume of 100 μl. Reactions are initiated by the addition of re¬ combinant Rl protein, and the assay mixture is incubated at 37°C for about 5-15 minutes for initial rate determinations. Reactions are quenched by immersion in a boiling water bath for 4 minutes . Samples are frozen and lyophilized to dryness . Lyophilized samples are reconstituted in a buffer at a basic pH containing Tris and Mg(0Ac)₂. Samples are centrifuged at high speed to precipitate denatured protein. The supernatant is then loaded onto columns of aminophenylboronate which had been pre-equilibrated with the same buffer. Deoxyribonucleoside diphosphates (dCDP or dGDP) are then eluted prior to the unreacted ribonucleoside diphosphate substrate and resolved from the substrate. Unreacted ribonucleoside diphosphate substrates can be quantitatively recovered and columns can be regenerated by treatment with sodium citrate, at pH 5.9. Radioactivity in aliquots of both the buffer B and citrate fractions can be determined by liquid scintillation counting.

B. Recombinant Pf R2 Activity Assay

Pf R2 is reactivated by iron and tyrosyl radial regeneration using the procedures described by Mann, et al. , Biochem. 30 1939-1947 (1991)) . After reactivation, ribo¬ nucleotide reductase/Pf R2 activity is determined from the rate of reduction of [³H] -CDP. One unit is defined as the amount of protein which, in the presence of excess the Pf Rl subunit, catalyzes the formation of 1 nmol of dCDP/minute at 37⁰C (Engstrόm et al.. Biochem.. 18 2941-2948 (1979) ; Ingemarson et al. , J. Virol. 63 3769-3776 (1989)) . The reactivated Pf R2 protein is assayed in the presence of 15 μg of pure Pf Rl protein, obtained as described above.

Chromosomal Location of Plasmodium Falciparum

Chromosomal sized DNA from Pf was isolated from agarose gel blocks, which had been subjected to electro¬ phoresis. Gels were run on a CHEF apparatus photographed and transferred to a solid support, e.g. , ZetaProbe™ (BioRad Laboratories, Richmond, CA) following the manufacturers recom¬ mended procedures to obtain a blot. Radioisotope-labelled probes complementary to Pf Rl and R2 DNA were designed to probe the blots for Pf Rl and R2 DNA. A radioisotope-labelled chromosome 14 control probe complementary to the DNA of a gene known to be on chromosome 14 was used as a control. A Pf chromosomal-sized DNA blot hybridized to all three of the probes, i.e., Rl, R2, and GPI probes. Thus , the chromosome 14 control probe verifies that genes coding for Rl and R2 are on chromosome 14. Synthesis and Purification of Antimalarial Compounds

Peptide intermediate portions of antimalarial compounds according to the present invention may be syn¬ thesized and purified by standard methods as outlined below. The peptide intermediates may be synthesized in vitro by chemical coupling and end-product purification methods well known to the ordinary practitioner in this art. (Climent, et al . , Biochem 30 5164-5171 (1991) ; and Bushweller et. al • , Biochem 30 8144-8151 (1991)) . Peptides according to the in- vention may be prepared using conventional solid phase synthesis procedures, such as those described by Merrifield, J. Am. Chem. Soc. 85:2149 (1964) . Other equivalent chemical syntheses known in the art can also be used, such as the synthesis of Houghten, Proc. Natl. Acad. Sci. 82:5132 (1985) .

Other examples of acceptable peptide synthesis techniques may be found in M. Bodanszky et al . , Peptide Synthesis. John Wiley & Sons, 2d Ed. (1976) . Other peptide synthesis procedures are well-known to those skilled in the art. A summary of peptide synthesis techniques may be found in J.Stuart and J.D. Young, Solid Phase Peptide Synthesis. Pierce Chemical Company, Rockford, IL (1984) . The synthesis of peptides by solutions methods may also be used, as described in The Proteins, vol. II, 3rd. Ed., Neurath, H. e_t al. , Eds., p. 105-237, Academic Press, New York, NY (1976) . Appropriate protective groups for use in such syntheses can be found in the above texts or in J.F.W. McOmie, Protective Groups in Organic Chemistry. Plenum Press, New York, NY (1973) . In general, these synthetic methods involve the sequential addition of one or more amino acid residues or suitably protected amino acid residues to a growing peptide chain. Normally, the amino group and, if relevant, the side chain, of each amino acid residue is protected by a suitable, selectively-removable protecting group. A different, selectively removable protecting group is utilized for amino acids containing a reactive side group, such as lysine.

In particular, polypeptides according to the present invention may be prepared to about 99% purity from (Fmoc) -amino acids on a Milligen 9600 synthesizer by automated solid-phase methods (^"Chang et al. , Int. J. Peptide Protein Res. 11246-249 (1978) ; Meienhofer et al. , Int. J. Peptide Protein Res. 1335-42 (1979) ) . Peptide coupling is obtained via diisopropyl carbodi-imide/hydroxybenzotriazole chemistry in a solvent such as dimethyl-formamide (DMF) . Fmoc groups are cleaved in a solvent such as piperidine/toluene/DMF. After removal of the final Fmoc group, the peptide is acylated. Deprotection of amino acid side chains and cleavage of the peptide from the resin is performed in trifluoroacetic acid/dimethylsulfide. The peptide can then be purified by liquid chromatography.

It is contemplated that, based upon the carboxy terminal amino acid sequence of Pf R2, peptide analogs or mimics may be prepared, blocked, and effectively screened for their abilityto selectively inhibit Pf RR according to assays described later herein. It is particularly contemplated that conservative amino acid changes may be made which do not alter the biological function of the polypeptide derivative. When conservative substitutions destroy the biological function of a peptide, this can be detected by screening the peptide for its ability to selectively inhibit Pf RR. Examples of conservative changes include for instance, substituting one polar amino acid, such as threonine, for another polar amino acid such as serine; substituting one acidic amino acid, such as aspartic acid for another acidic amino acid, such as glu- tamic acid; substituting a basic amino acid, such as lysine, arginine or histidine for another basic amino acid; or substituting a non-polar amino acid, such as alanine, leucine or isoleucine for another non-polar amino acid.

Crude peptides may be injected into an analytical HPLC column to optimize the purification. Following elution, a sample load of 20 mg per injection may be chromatographed on a semi-preparative C₁₈ column such as a Dychrom 1 cm x 25 cm HPLC column using a binary solvent system such as 0.1% trifluoroacetic acid (TFA) and 0.1% TFA in acetonitrile, and appropriate gradients. Peptide elution may be monitored at 214 nM. HPLC fractions may be collected, lyophilized and analyzed by mass spectrometry using fast atomic bombardment (FAB) conditions on instruments such as VG ZAB E.

The amino-blocked peptides of the invention are prepared from polypeptide intermnediates according to the peptide N-terminal blocking method previously described by Yang et al. , FEBS Lett. 272 61-64 (1990), for example.

Antimalarial Pharmaceutical Compositions Antimalarial compositions are provided which comprise a pharmaceutically acceptable carrier and at least one compound according to the invention which has a length of from 7 to about 20 amino acids which inhibits P.falciparum ribonucleotide reductase catalyzed reduction of ribonucle- otides to 2' -deoxyribonucleotides. The antimalarial peptide derivatives may be combinedwith anypharmaceutically acceptable carrier suitable forparenteral, preferablyintravenous administration. Thus, the antimalarial compounds may be formulated according to conventional methods for preparing peptide agents or blocked peptide agents for parenteral administration. The optimum concentration of the antimalarial blocked peptides in the chosen medium can be determined empirically, according to procedures well known to medicinal chemists. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, and other ingredients which may be appropriate for the desired route of administration of the pharmaceutical peptide preparation. The use of such media for pharmaceutically active substances is known in the art. Any conventional media or agent is contemplated for use in the pharmaceutical preparation according to the invention insofar as it is compatible with the antimalarial peptides according to the invention.

The intravenous carrier most advantageously comprises a pharmaceutically acceptable buffer solution such as phosphate buffered saline at physiological pH, preferably in combination with a pharmaceutically acceptable compound for adjusting isotonic pressure, such as mannitol or sorbitol and similar substances. The antimalarial composition may be tested for its ability to inhibit Pf RR reduction of ribonucleotides to 2' - deoxyribonucleotides via a Pf RR activity assay. The antimalarial composition is added to a recombinant Pf RR enzyme solution containing labelled ribonucleotides. For comparison a control solution is run under the same condi¬ tions, but without the anti-malarial composition being present. Both solutions are incubated, followed by assaying to determine the amount of ribonucleotides that remain. Less ribonucleotides remaining in the control solution indicates that the antimalarial composition will inhibit the Pf RR enzyme activity.

Method for Treating Malaria Compounds according to the present invention having a length of from 7 to about 20 amino acids may be administered by any convenient route which will result in the delivery to the bloodstream of an antimalarial effective amount thereof. Contemplated routes of administration include both parenteral and oral routes. Intravenous administration is presently contemplated as the preferred administration route.

Generally, a peptide whose N-terminus is optionally blocked may be administered in an amount sufficient to obtain the desired therapeutic effect. The compounds of the inventionmaybe administered at appropriate intervals, until the danger or symptoms of the disease are no longer evident, after which the dosage may be reduced to a maintenance level or discontinued.

As used herein, the term "patient" includes both humans and animals. The inhibition of growth and replication of the malarial organisms in red blood cells (erythrocytes) is monitored at intervals after admin-istration to evaluate the peptide potency and clearance rates. The blood samples are also evaluated for growth and reproduction of malarial organisms, e.g.. by isolation andmeasurement of the malarial organism' s mRNA or by probing for the presence of either a Pf Rl or a Pf R2 protein with a monoclonal antibody.

After evaluation, the compound dosage may be ad¬ justed as necessary to obtain or maintain inhibited growth and replication of malarial organisms. In the weeks that follow, the patient is again monitored once or twice weekly, until the patient is free of disease symptoms. Ordinarily, the patient is treated until all red blood cells examined are free of malarial organisms. Thus, the patient should be treated until the red blood cells containing malarial organisms die and new cells develop which are free of malarial organisms. In areas with a high risk of malarial infection, indefinite daily administration of a therapeutic dosage may be necessary to avoid an initial infection or prevent a reinfection.

Method for Detecting a Malarial Parasite in a Patient

The presence of the malarial parasite in the patient's blood can be detected by a diagnostic polymerase chain reaction (PCR) assay. In principle any two regions of parasite DNA that show sequence divergence from the host can be used for diagnostic PCR. For example, one primer can be synthesized to have a nucleotide sequence corresponding to the region of DNA coding for Pf R2 amino acid numbers 273-278. The other primer will be complementary to a nucleotide sequence coding for the Pf R2 C-terminal region (see SEQ ID NO:2) . Examples of P. falciparum primers are 5' -ATT TTC CAT TCC AAA-3' (SEQ ID NO:41) and 5'-AAA TTC CGT ATT CAG ACA-3' (SEQ ID NO:42) , which are upstream primer and downstream primer, respectively. Diagnostic PCR can be carried out by isolation of DNA from blood samples obtained via peripheral circulation using a variety of published methods, one of which is described below. PCR can be carried out using lμM of each primer above in standard PCR buffer. The above example of PCR primer pairs would amplify a fragment of Pf DNA 196 base pairs in length,

A sample containing red blood cells is obtained from a patient suspected of being infected with malarial parasites. The red blood cells are collected by centri¬ fugation and rinsed with phosphate buffered saline (PBS) until about 6 milliliters of RBCs are obtained. The RBCs are resuspended in PBS. Saponin is added followed by incubating to release the suspected parasites. The samples suspected of having parasites are rinsed with PBS, resuspended in lysis buffer and incubated "at room temperature. The solution is adjusted with RNase and protease incubated at 37°C to digest the RNA and protein present. Digested proteins are precipi- tated by addition of a salt such as NaCl. The DNA is then extracted with a phenol/chloroform solvent, precipitated with ethanol, washed and resuspended in water.

Adiagnostic polymerase chain reaction (PCR) assay is performed on the resuspended DNA. Primers complementary to any two regions of parasitic DNA corresponding to a portion of either SEQ ID NO:l or SEQ ID NO:3 that showed divergence from the hosts Rl and R2 can be used for the diagnostic PCR. Analysis can be performed on approximately 10% of the sample by running the sample portion on 8.0% polyacrylamide. The primers should amplify a fragment of DNAabout 196 nucleotides in length. Gels can be photographed and transferred to a solid support, e.g., ZetaProbe™ (BioRad Laboratories, Richmond, CA) , following the manufacturer's recommended procedures to obtain a blot. Radioisotope-labelledprobes complementary to the amplified portion of the PF Rl or R2 DNA can be designed to probe the blots for the parasitic DNA. Probe binding would indicate the presence of parasitic DNA. A blood sample known to be infected by the parasite can be utilized as a control and run through the same procedures as described above to verify the diagnosis.

Antimalarial Peptides and Polypeptide Segments of Pf Rl

The antimalarial polypeptides according to the invention are described as follows.

Attachment of a peptide to an Pf Rl polypeptide at the Pf R1/R2 binding site inhibits the formation of an active Pf R1/R2 complex. Pf R2-derived peptides according to the present invention display much greater binding affinity toward Pf Rl than toward a mammalian Rl enzyme. For example, the blocked peptide of Example 4 below has an IC₅₀ of 160 μM for binding mammalian Rl. Because the present peptides preferentially bind Pf Rl, substantially all the available Pf Rl can be bound by peptides, while leaving the mammalian host Rl free for association with host R2 and formation of the active host RR holoenzyme. This therapeutic effect is obtained by starting at an effective amount of a short antimalarial peptide based upon the in vitro Pf RR inhibition assay and adjusting the dosage (if necessary) until inhibition of the formation of the Pf RR holoenzyme is observed.

Thus, the blocked peptides according to the present invention, are selective inhibitors of Pf RR and are thera- peutically useful antimalarial agents and may be selective inhibitors of other related protozoan RRs. Eucaryotic R2 polypeptides previously characterized have been highly homologous and have demonstrated considerable cross-reactivity with RRs of other eucaryotic species. For example, the twenty C-terminal residues of mouse and human R2 differ by only 10% (2/20) . The homology is even closer for the seven C-terminal residues. The human R2 seven C-terminal amino acids are identical to those of other eucaryotic organisms such as clam and mouse.

Although Pf is a eukaryote, Pf R2 is less homolo- gous with mammalian R2. The amino acid sequence of the Pf R2 subunit is 65% different (13/20 different amino acid positions) from mouse R2 in the twenty C-terminal residues. Moreover, the amino acid sequence of the Pf R2 subunit is 43% different from the human R2 in the seven C-terminal positions, with only three of the seven (3/7) human amino acid positions being identical to Pf R2. The conserved amino acids are underlined.

(SEQ ID NO:6) Phe-Thr-Leu-Asp-Ala-Asp-Phe (Human, Mouse and Clam) , and (SEQ ID NO:7) Phe-Cys-Leu-Asn-Thr-Glu-Phe

(P. falciparum) .

Table 1, below, lists for comparative purposes C- terminal amino acid R2 sequences for various organisms. TABLE 1 C-TERMINAL SEQUENCES FOR R2

ORGANISM SEOUENCE

E. coli SEQ ID NO:9 EVDTDDLSNF QL

Clam SEQ ID NO:10 GGNTGDSHA. FTLDADF

Yeast SEQ ID NO:11 KSTKQEAGA. FTFNEDF

Mouse SEQ ID NO:12 NSTENS FTLDADF

Human SEQ ID NO:13

..TENS.... FTLDADF

Vaccinia SEQ ID NO:14 QEDNH FSLDVDF

Herpes SEQ ID NO:15 TSYAGAWND L

Varicella SEQ ID NO:16 TSYAGTVIND L

The practice of the invention is illustrated by the following non-limiting examples.

Example 1 Isolation of DNA from Plasmodium Falciparum

Parasites were maintained and stored as de-scribed by Taraschi, et al.. Science 232102-104 (1986) . Genomic DNA was purified essentially as described by Medrano, et al.. Biotechnigues 8., 43-45 (1990) with the following modifica- tions. Infected red blood cells (RBCs) from 10 plates were collected by centrifugation (room temperature, 9 minutes) and rinsed with phosphate buf-fered saline (PBS) . The packed, infected RBCs (6 ml) were re-suspended in 30 ml of PBS. Saponin was added until a concentration of 0.1% was obtained, followedby incubating for 5 minutes to release the parasites.

Freed parasites were rinsed with 30 ml PBS, re¬ suspended in 15 ml lysis buffer (2.5 mM Tris.HCl at pH 8.0, 5 mM EDTA, 4% sodium lauryl sarcosinate (Sarkosyl™) ) and incubated for 5 minutes at room temperature. The solution was adjusted to 50 μg/ml RNase and 100 μg/ml of a subtilisin- type protease (e.g.. Proteinase K) and incubated at 37°C for 2 hours. Proteins were precipitated by the addition of 5 ml of 5 M NaCl at 4°C for 12 hours. The solution was then extracted with an equal volume of phenol/chloroform. The DNA was precipitated with ethanol, washed and re-suspended in water overnight at 37°C.

Polymerase chain reaction (PCR) primers were designed to amplify Rl and R2 DNA. Samples containing 1 μg of total genomic DNA obtained from Pf as described above, were amplified via PCR with 2 μM of S.N2 (SEQ ID NO:17) and S.C2 (SEQ ID NO:18) primers. The PCR procedure was performed in a 100 μl volume containing 50 mM KCl, lOmM Tris.HCl pH=8.0, 2 mM MgCl₂, 2 mM each dNTP and 2.5 units Tag DNA polymerase (e.g.. Amplitaq™, Perkin-Elmer, Norwalk, CT) . Samples were subjected to 35 cycles of PCR, performed on an Ericomp thermal cycler. Each cycle was 94°C, 30 seconds; 40°C, 60 seconds; and 72°C, 90 seconds. After amplification, the DNA was made blunt-ended with T4 polymerase, kinased with T4 polynucleotide kinase and ligated to Eco RV-digested KS (+) plasmid (Stratagene, La Jolla, CA) . The recombinant plasmids were isolated by standard techniques and sequences determined by the chain termination reaction with a modified T7 bacterio- phage DNA polymerase (e.g. , Sequenase™ (United States Biochemical Corporation, (USB) ) ) .

The Pf R2 N-terminal nucleotide sequence was isolated by anchored PCR (APCR) . This genomic DNA was digest¬ ed with Xbal. Fragments of 600-850 base pairs (bps) in length were gel purified from a 1% agarose gel. seventy nanograms of size-selected DNA from the agarose gel was ligated to 240 ng of Xbal, 4-chloro-3-indolyl phosphate (CIP) treated KS+ in a 100 μl reaction volume. Seven μl samples of the ligation mix were amplified with S.C5 (SEQ ID NO:19) and either the universal forward or reverse primers. PCR conditions were 94°C, 15 seconds; 35°C, 30 seconds; and 72°C, 90 seconds. The resulting DNA fragment was gel purified, re-amplified and directly sequencedvia the procedures set forth in Thein, Com¬ ments (USB Technical Guide) .16. 8 (1989) . The Pf R2 C-terminal nucleotide sequence was isolated by forced cloning and screening of colonies . Genomic DNA (obtained as described above) was digested with Asel and Xbal enzymes. Fragments of 750-900 bps length were size- selected from a 0.7% agarose gel. A Ndel site was inserted in EcoRl site of Bluescript KS+ (termed KS-Ndel) via Ndel- EcoRl linkers. One hundred μg of the size-selected, digested DNA was ligated into 50 μg Bluescript KS-Ndel digested with Xbal and Ndel. DH5α E. coli cells were transformed with the ligation mix. Colonies were screened for positive clones by standard procedures with a 140 bps fragment of R2 amplified from high molecular weight DNA with S.C8 (SEQ ID NO:20) and S.N9 (SEQ ID NO:21) primers. The positive clones were isolated and lysed to release the plasmids. The plasmids were digested to yield Pf DNA fragments. The resulting DNA frag¬ ments were sequenced in the same manner described above for the N-terminal segments.

The entire Pf R2 genomic DNA sequence is set forth as SEQ ID NO:l.

Example 2

Isolation and Sequencing of Gene Encoding for Plasmodium Falciparum Rl

Genomic Plasmodium falciparum DNA was obtained as in Example 1, above, and amplified by a PCR procedure. The PCR solution contained 1 μg of genomic DNA, and 1 μM L.N2 (SEQ ID NO:22) and L.C4 (SEQ ID NO:23) primers. The PCR procedure was performed in a 100 μl sample volume containing 50 mM KCl, 10 mM Tris.HCl pH = 8, 2 mM MgCl₂, 2 mM each dNTP and 2.5 units of a Taq DNA polymerase, Amplitaq™ (Perkin-Elmer, Norwalk, CT) . Samples were subjected to 35 cycles of PCR, performed on an Ericomp thermal cycler as set forth in Example 1. Each cycle was 94°C, 30 seconds; 37°C, 60 seconds; and 72°C, 60 seconds. Extension of the Rl clone was accom¬ plished with a Pstl inverted PCR reaction. One hundred μg of Pstl digested Pf DNA was self-ligated in a 40 μl volume. The entire ligation mix was subject to PCR in a 100 μl volume with L.N3 (SEQ ID NO:24) and L.C5 (SEQ ID NO:25) primers. Amplification conditions were 15 cycles (at 94°C, 30 seconds; 40°C, 30 seconds; and 72°C, 90 seconds) followed by 20 cycles (94°C, 30 seconds; 50°C, 30 seconds; and 72°C, 90 seconds) . Segments of Rl were isolated and sequenced as set forth in Example 1. The complete DNA sequence for the Rl gene is set forth as SEQ ID NO:3. The Pf Rl polypeptide encoded by the DNA sequence according to SEQ ID NO:3 is set forth as SEQ ID NO:4.

Example 3 Preparation and Purification of Ac- (SEQ ID NO:7)

The acylated polypeptide Ac- (SEQ ID NO:7) was prepared on a Milligen 9600 synthesizer with the Fmoc procedure of Chang, et al. , Intl. J. Pep, and Prot. Res. 11 246-249 (1978) as described in the standard Milligen protocol. Peptide coupling was achieved via diisopropyl carbodiimide/hydroxybenzotriazole chemistry in dimethyl- formamide (DMF) . Fmoc groups were cleaved in piperi- dine/toluene/DMF. After removal of the final Fmoc group, the peptide was acetylated with 5% acetic anhydride/2.5% diisopropyl ethylamine. Deprotection of amino acid side chains and cleavage of the peptide from the resin was performed in trifluoroacetic acid/dimethylsulfide or trifluoroacetic acid /l, 2-ethanedithiol.

The crude peptide was initially injected onto an analytical HPLC column to optimize the purification procedure. Then, a sample load of 10-20 mg per injection was chromatog- raphed on a semi-preparative Dychrom 1 cm x 25 cm reversed phase HPLC column using a binary solvent system, consisting of 0.1% TFA and 0.1% TFA in acetonitrile, and appropriate gradients. Peptide elution was monitored at 214 nM and 258 nM. HPLC fractions were collected, lyophilized and then analyzed by mass spectrometry using FAB conditions on a VG ZAB E instrument. A mass spectrum of purified Ac- (SEQ ID NO:7) peptide showed a dominant molecular ion of 915, corresponding to an M + H peak. ^**HNMR (ID,2D COSY, TOCSY) , mass spectra, and amino acid analysis were fully in accord with the assigned structure for this acylated heptapeptide (Tables 2 and 3, below) . Table 2 Random Coil ^**H Chemical Shifts for Ac- (FCLNTEF) Residue ^**H Chemical Shift (ppm) Protons

Phe acetyl methyl 1.7 CH₃

Leu 0.8 δCH₃ 1 ( 6H)

1.4 |8CH₂

1.6 γCH

Thr 1.0 γCH₃ 0.4 /3CH

Glu 2.2 ΎCH₂

1.9 SCH₂ Phe, Asn, Cys 2.4-3.1 |8CH₂ ( 8H)

Backbone methines 4.1-4.6 αCH ( 7H) Asn 6.9-7.4 γNH₂

Phe aromatics 7.1-7.3 ring ( IOH:

Amides 7.6-8.3 αNH ( 7H:

Table 3

Amino Acid Analysis of Ac- (FCLNTEF)

Amino Acid No. Residues Found

Asx 1.05 Glx 1.01

Thr 1.00

CSSC 0.51

Leu 1.00

Phe 2.00

Examples 4-53

Preparation and Purification of Polyeptides

Polypeptides corresponding to SEQ ID NOS: 5, 6,

8-16, 26-39, 43-67, and N-terminally acylated derivatives were produced in the same manner as in Example 3, above. Example 54

Peptide Inhibition of Calf Mammalian Ribonucleotide Reductase Activity. The inhibitory effects of acylated and non- acylated polypeptides on mammalian RR enzymatic activity was assayed as described in Yang, et al . , FEBS Lett. 272 61-64 (1990) ) .

Calf thymus Rl was obtained and purified as de- scribed Yang, et al.. id. , and recombinant mouse R2 was ob¬ tained andpurified from transfected E. coli via an expression vector (See, e.g.. Mann, et al. Biochem. 30, 1939-1947 (1991)) . The RR activity resulting from combining these purified mammalian Rl and R2 subunits was verified as described in Yang, et al.

As a control, the purified Rl was first exposed to varying amounts of an acylated heptapeptide corresponding to the 7 amino acid C-terminal sequence of mouse and human R2 (Ac- (SEQ ID NO:6), obtained by the procedure in example 3, above. After the the Ac- (SEQ ID NO:6) had sufficient time to bind with the Rl, the purified recombinant mouse R2 was added to the mixture, and the mixture assayed for RR activity. An IC₅₀ of 10-20 μM was observed. Using the same procedure, an IC₅₀ of 160 μM was observed for Ac- (SEQ ID NO:7) The inventive peptide Ac- (SEQ ID NO:7) was thus about 8-16 times less potent an inhibitor on mammalian RR activity than Ac- (SEQ ID NO:6), which has an amino acid sequence identical to the seven C-terminal residues of mouse/human R2. Accordingly, Ac- (SEQ ID NO:7) has a surprisingly lower affinity for mammalian R2 than Ac- (SEQ ID NO:6) .

Table 4 below shows the inhibitory potency of other acylated and unacylated peptides as compared to SEQ ID NO:6 toward mammalian RR activity.

TABLE 4

Peptide IC^'.μM Percent Rlb

1. Ac- (NSFTLDADF) 9-15 100

AC- (SEQ ID NO:43) Ac- (SFTLDADF) 8-20 100 Ac- (SEQ ID NO:44)

SFTLDADF 80 25

(SEQ ID NO:44)

AC- (FTLDADF) 8-20 100 AC- (SEQ ID NO:6)

FTLDADF >400^d <3

(SEQ ID NO:6)

^alC₅₀ is the concentration of peptide producing 50% inhibition of activity obtined in the absence of peptide.

Values are derived from Dixon plots of 4-6 concentration points. From two to four assays were run for each point.

Range of IC_S0 values are for two independent determi-nations conducted on different days.

"Percent relative inhibitory potency, defined as 100 x ( (IC₅₀ Ac- (FTLDADF) / (IC₅₀ peptide analog) ) .

TABLE 4 (Cont.l)

Peptide IC₅₀".μM Percent RI^b

Ac- TLDADF) >400 <3 Ac- SEQ ID NO:45)

Ac- LDADF) >400 <3 Ac- SEQ ID NO:46)

8. Ac- LTLDADF) >400 <3 Ac- SEQ ID NO:47) 9. Ac- F(4' -NH,)TLDADF) 100 10 Ac- F(4'-NH₂)- (SEQ ID NO:45))

10 Ac- F(4'-N₃)TLDADF) 100 10 Ac- F(4' -N₃) - (SEQ ID NO:45))

11, Ac- FSLDADF) 40-42 25 ± 1.1 Ac- SEQ ID NO:48)

12. Ac- FALDADF) 35-40 24 ± 4.1 Ac- SEQ ID NO:49)

13. Ac- FTVDADF) 30 35 Ac- SEQ ID NO:50) 14, Ac- FTFTADF) 48-58 18 ± 4.2 Ac- SEQ ID NO:51)

15. Ac- FTADADF) 207 4.9 + 0.60 Ac- SEQ ID NO:52)

16, Ac- FTLNADF) 25-29 40 ± 2.4 Ac- SEQ ID NO:26)

17. Ac- FTLAADF) 286-336 2.9 + 0.4 Ac- SEQ ID NO:53)

18 Ac- FTLDGDF) 110-230 9 + 0.6 Ac- SEQ ID NO:54) 19. Ac- FTLDLDF) 28 35 Ac- SEQ ID NO:55) TABLE 4 (Cont.2)

Peptide IC₅₀ ^**,μM Percent RI^**

20. Ac- FTLDAEF) 450-620 1.7 ± 0.15 Ac- SEQ ID NO:56)

21. AC* FTLDANF) 32-38 23 ± 1.0 AC* SEQ ID NO:57)

22. AC- FTLDALF) 29-30 35 ± 5.0 AC- SEQ ID NO:58)

23. Ac- FTLDAAF) 34-35 28 ± 1.3 AC- SEQ ID NO:59)

24. AC- FTLDADL) >400 <3 Ac- SEQ ID NO:60)

25, AC- FLDADF) (6 pos. del.) 412 2.5 AC- SEQ ID NO:61)

26 Ac- FTLDDF) (3 pos. del.) 322 Ac- SEQ ID NO:62)

27, Ac- FTLDF) (3 & 4 pos. del.) 364-460 2.5 ± 0.18 Ac- SEQ ID NO:63)

28 Ac- FTLDADFAA) 500 2.1 Ac- SEQ ID NO:64)

Example 55

Inhibition of Mammalian and Yeast RRs

The same RR assay was conducted as in Example 54, but substituting S. cerevisiae (yeast) RR. A mammalian RR assay was conducted. Table 5 below shows the comparative inhibitory potency results against yeast and mammalian RR for four acylated polypeptides having an amino acid sequence a- ccording to SEQ ID NO:65-67 and 6, respectively. TABLE 5

Peptide IC^'.uM IC_^'.uM

Yeast RR Mammalian RR

29. Ac- (AGAFTFNEDF) 25 Ac- (SEQ ID NO:65)

30. AC-(FTFNEDF) 25 100 Ac- (SEQ ID NO:66)

31. Ac- (TFNEDF) 600 600 Ac- (SEQ ID NO:67) 4. Ac- (FTLDADF) 25 8-20

Ac- (SEQ ID NO:6)

^aIC₅₀ is the concentration of peptide produing 50% inhibition of activity obtined in the absence of peptide.

Values are derived fromn Dixon plots of 4-6 concentration points. From two to four assays were run for each point.

Range of IC₅₀ values are for two independent determi-nations conducted on different days.

Example 56

Recombinant Production of the Pf Rl Subunit

The Pf DNA segment that codes for the Pf Rl subunit is cloned in baculovirus system by following cloning procedures essentially as described by Salem, et al . , FEBS, 323. No. 1,2, 93-95 (1993), as follows.

A. Obtaining and Amplifying a DNA Segment Coding for the Pf Rl subunit

Pf Rl DNA, having a sequence according to SEQ ID

NO:3, is obtained from parasites that are maintained and stored as described by Taraschi, et al . Science 232 102-104

(1986) . Genomic DNA is purified essentially as described by Medrano, et al. , Biotechnigues 8., 43-45 (1990) with the following modifications. Infected red blood cells (RBCs) from

10 plates are collected by centrifugation (room temperature,

9 minutes) and rinsed with phosphate buffered saline (PBS) .

The packed, infected RBCs (6 ml) are resuspended in 30 ml of PBS. Saponin is added until a concentration of 0.1% is obtained, followed by incubating for 5 minutes to release the parasites .

Freed parasites are rinsed with 30 ml PBS, resuspended in 15 ml lysis buffer (2.5 mM Tris.HCl at pH 8.0, 5 mM EDTA, 4% sodium lauryl sarcosinate (Sarkosyl™) ) and incubated for 5 minutes at room temper-ature. The solution is adjusted to 50 μg/ml RNase and 100 μg/ml of a subtilisin type protease (e.g. , Proteinase K) and incubated at 37°C for 2 hours. Proteins are precipitated by the addition of 5 ml of 5 M NaCl at 4°C for 12 hours. The DNA is then extracted with an equal volume of phenol/chloroform, is precipitated with ethanol, and is washed and resuspended in water overnight at 37°C. Polymerase chain reaction (PCR) primer pairs complementary to the N-terminus and C-terminus, respectively, of SEQ ID NO:3 or SEQ ID NO:40, which primers terminate with the Nhel restriction site, are designed to amplify Pf Rl DNA and add the Nhel restriction site to each end of the Pf Rl DNA segment. Samples containing 1 μg of total genomic DNA obtained from Pf as described above (or about 0.1 μg of the synthetic Pf Rl DNA segment according to SEQ ID NO:40) , are amplified via PCR with 2 μM of the PCR primers. The PCR pro¬ cedure is performed in a 100 μl volume containing 50 mM KCl, lOmM Tris.HCl pH=8.0, 2 mM MgCl₂, 2 mM each dNTP and 2.5 units TagDNA polymerase (e.g.. Amplitaq™, Perkin-Elmer) . Samples are subjected to 35 cycles of PCR, which is performed on an Ericomp thermal cycler. Each cycle is 94°C, 30 seconds; 40°C, 60 seconds; and 72°C, 90 seconds. Alternatively, a Pf Rl DNA segment is synthesized which has a sequence according to SEQ ID NO:40. Such synthesis is performed using DNA organic chemical synthetic methods well known to one of ordinary skill in the art.

B. Construction of the pBlueBac2-Pf Rl Vector

The amplification product from the above ampli¬ fication process is digested with Nhel and inserted into the Nhel site of pBlueBac2 (InVirtrogen) to produce a Pf Rl vector (pBlueBac2-Pf Rl) . Spodoptera frugiperda (Sf9) cells are transformed with the recombinant plasmid using the cationic liposome method (Hartig et al. , Biotech. , 11, 310-313 (1991)) . Positive clones are selected by beta-galactosidase blue/white screening and subsequently grown in 3 ml volumes in a 24-well dish. Plaques are subjected to PCR analysis with primers complementary to the polyhedron loci to select those free of non-recombinant baculovirus.

C. Growth and Maintenance of Sf9 Cells Sf9 cells are cultured at 27°C in Excell 400 medium

(JRH Scientific) supplemented with 10% heat inactivated Fetal Calf Serum (FCS) (BRL) , 540 μg/ml gentamycin and 2.5 μg/ml Fungizone that has been sterile filtered. Cells grown in spinner flasks are also supplemented with 0.1% Pluronic F-68 (JHR Scientific) to reduce shear damage.

D. Expression of Recombinant Pf Rl

Original stocks of recombinant virus, produced as above, are used to infect virgin Sf9 cells to screen for protein production and generate the second generation of virus. Approximately 2 x IO⁶ cells are adhered to each well of a 24-well dish in a 3 ml volume and infected with 20, 100 and 500 μl of recombinant virus. Aliquots are removed and assayed for expression of Pf Rl every 24 h for 4 days. Plaques producing Pf Rl protein without viral contamination of the protein are selected for further study. For large scale expression of the Pf Rl protein, 50 ml of cells are grown to a density of 1.3 x IO⁶ cells/ml and infected with 10⁸ virus particles. Cells are grown for 60-72 hours then har¬ vested by centrifugation at 1,000 x G for 10 minutes.

E. Purification of Recombinant Pf Rl

The cell pellet collected from a 50 ml culture is resuspended in 5 ml of lysis buffer (50 mM Tris-Cl, pH 7.6, 0.1 mM DTT, 2mM PMSF) and subjected to two cycles of freeze- thaw. The lysate is centrifuged at 105,000 x G for 30 minutes. The resulting supernatant is loaded directly onto an FCLNTEF-Sepharose (FCLNTEF = SEQ ID NO:7) affinity column using the method described in Yang, et al . , FEBS Lett. 272 61-64 (1990)) . The column is washed with 50 mM Tris-Cl, 0.1 mM DTT (buffer A) and then with buffer A + 100 mM KCl. Rl is eluted with 10 ml buffer A + 500 mM KCl. The column is regenerated by treatment with 6 M guanidine-HCl. Column fractions are monitored by the Bradford assay (Bradford, Anal. Biochem. 72 248-254 (1976) .

F. Pf Rl Identification

A partial N-terminal sequence of the purified protein is determined as in the process of Edman, et al . Eur. J. Biochem. 180-91 (1967) using an Applied Bio-systems model 473A sequencer. SDS polyacrylamide gel electrophoresis (SDS- PAGE) is performed using 7.5% acrylamide. Western blots are performed as described in Rubin et al . J. Biol. Chem. 265, 1199-1207 (1990) using a monoclonal antibody raised against a synthetic protein segment of Pf Rl which is unique to Pf Rl as obtained from SEQ ID NO:3. Production of monoclonal antibodies (MABs) specific for regions of Pf Rl such as the Pf R2 binding site is well within the skill of the ordinary practitioner in the art. For example, MABs are raised to Pf Rl and screened to determine whether binding of Pf R2 it Pf Rl is blocked. Such a MAB would be specific for Pf Rl. Protein concentration is determined according to the Bradford assay (Bradford, Anal. Biochem. 72 248-254 (1976) .

Example 57 Recombinant Production of the Pf R2 Subunit

Expression of recombinant Pf R2 is based on transcription of T7 RNA polymerase according to the method of Mann, et al . , Biochem. 30 1939-1947 (1991)) . The Pf R2 protein is expressed and purified to homogeneity following a rapid and simple purification procedure. The Pf R2 protein is expressed from T7 RNA polymerase responsive plasmids, which are constructed by using standard molecular cloning tech¬ niques. Plasmids are propagated in E. coli strain BL21(DE 3) grown in LB medium at 37°C, in the presence of carbeni- cillin, 50 μg/mL. Transfections are performed according to the method of Hanahan, J. Mol . Biol. 166 557-580 (1983) .

A. Obtaining and Amplifying a DNA Segment Coding for the Pf R2 Subunit

Total Pf DNA comprising the Pf R2 DNA segment, having a sequence according to SEQ ID NO:l, and coding for

Pf R2, is first obtained from parasites in the same manner as for the Pf Rl segment in Example 56, above. PCR primer pairs terminated with a Nhel restriction site and complementary to the N-terminus and C-terminus of SEQ ID NO:l, respectively, are designed. The primers amplify the Pf R2 DNA segment having a sequence according to SEQ ID NO:l and add Nhel restriction sites to the ends of the Pf R2 DNA segment. The Pf R2 DNA segment is amplified by PCR in the same manner as for Pf Rl in Example 56, above.

B. Construction of the pET3a-Pf R2 Vector

The Pf R2 PCR amplification product is cleaved with Nhel restriction enzyme to produce a Nhel Pf R2 DNA fragment construct. This Pf R2 construct is transferred into the T7 expression vector pET3a (Rosenberg et al. , Gene 56 125-135 (1987) ; Studier et al .. Methods Enzvmol .. 185 60-89 (1990)) which is opened with the Nhel restriction enzyme. The resulting pET3a-Pf R2 vector is transfected into an E. coli strain BL21 (DE3) (Rosenberg et al . , Gene 56 125-135 (1987)) , which strain contains a lac (IPTG) -inducible, chromosomal copy of the T7 RNA poly-merase gene. The plasmid pET3a and the bacterial strain BL2KDE3) , are described in Studier et al . , Methods Enzvmol. , 185 60-89 (1990)) .

C. Growth and Maintenance of BL21(DES) Cells BL21 (DE3) E. coli bacteria transfected with the pET3a-Pf R2 plasmid are logarithmically grown in 3L of LB medium containing carbenicillin, 50 μg/mL. The LB medium is infected with 5 mL of overnight cultures of transfected BL21(DES) bacteria and cultured under agitation, (shaken 275 times per minute) at 37°C. The LB medium containing the transfected BL21 (DES) bacterial culture is supplemented with IPTG (0.5mM) when the absorption density at A₅₉₀ is 0.6-1.0. The transfected BL21(DE3) bacteria are then cultured for an additional four hours before harvesting.

D. Expression of Recombinant Pf R2 Protein

Pf R2 protein is produced by induction of log¬ arithmically growing BL21 (DE3) bacteria containing the plasmid pET3a-Pf R2 as described above. After incubation as described above, the cultures are chilled and centrifuged at 2500 G for 15 minutes at 2°C. The pellet from the first centrifugation is gently resuspended in 100-200 mL of 50mM Tris, pH 7.6 and 1 mM EDTA, and centrifuged at 1000 G for 10 minutes. The pellet from the second centrifugaion is frozen in liquid nitrogen and stored at -70°C.

E. Purification of Recombinant Pf R2 Protein Each purification protocol involves disinte-gration of the frozen BL21(DE3) bacteria pellet containing the Pf R2 protein, extraction into a buffer, precipitation of nucleic acids with streptomycin sulfate, ammonium sulfate precipita¬ tion, and anion-exchange chromatography. All operations are performed in a cold room at about +4°C.

Pf R2 preparation, which is a modification of the previously published procedure for purification and activation of recombinant E. coli R2 (Sjoberg et al. , Biochem. 2615658- 5662 (1986)), is described in detail where it will differ. Protein concentrations and purity may be assessed by Coomassie Brilliant Blue dye binding, with reference to a bovine serum albumin standard (Bradford, et al. , Anal. Biochem.. 72 148-254 (1976) ) , combined with laser densitometric (LKB Pharmacia) scanning of TCA-precipitated samples separated on 10% SDS- polyacrylamide gels (Engstrδm et al .. Biochem. , 182941-2948

(1979)) . Concentrations of highly purified proteins are measured by light absorbance and calculated from extinction coefficients, as discussed below.

Frozen bacterial pellets (W g) of BL2KDE3) E. coli bacteria containing plasmid pET3a-Pf R2 are obtained from centrifugation of the above cultures. The pellets are finely ground in a mortar with 2xW g of cold aluminum oxide (Sigma) , using liquid nitrogen as necessary to prevent thawing. The powder is either stored (-70°C) or directly mixed and thawed rapidly on ice in 4xW mL of extraction buffer (50mM Tris-HCl, pH 7.6, 1 mM PMSF, and 1 mM EDTA) and centrifuged for 40 minutes at 44000 G and 2°C. Streptomycin sulfate, 10% (w/v) , pH 7.0, is added to the supernatant, to a final concentration of 2.5% while stirring on ice, followed by centrifugation for 20 minutes at 27000 G and 2°C. Solid ammonium sulfate (0.243 g/mL, nominally 40%) is then added to the supernatant and the precipitate is recovered by centrifugation for 30 minutes at 27000 G and 2°C. After the precipitate is dissolved in extraction buffer, the extract is iron-reactivated (see Example 9B, below) or directly equilibrated (apo-Pf R2 preparations) in 50 mM Tris-HCl, pH 7.6, 1 mM PMSF, and 1 mM EDTA on a column containing Sephadex G-25 medium. Then about 30 mg of partially purified Pf R2 is loaded onto a 3 mL (3.8 x 1 cm) DEAE-cellulose column (DE 52, Whatman) , previously equilibrated with 10 mM potassium phosphate, pH 7.0, 30 mM KCl, and 1 mM EDTA. The column is washed with 10 mL of the same buffer and Pf R2 is then eluted in 3-4 column volume of 10 mM potassium phosphate, pH 7.0, 70 mM KCl, and 1 mM EDTA. The protein eluate is frozen directly at -70^oC or is recovered by overnight dialysis at 4°C against saturated ammonium sulfate. The eluated or dialysized protein is centrifuged for 30 minutes at 25000 G and 2°C, is dissolved in 50 mM Tris- HCl, pH 7.6, and is then stored in aliquots at -70°C.

Example 58 Enzymatic Activity of Recombinant Pf Rl and R2 A. Recombinant Pf RR Assays

Ribonucleotide reductase activity is assayed at 37°C using the method of Moore and Peterson, Biochem. 132904- 2907 (1974) with minor modifications . Briefly, assay mixtures contain 60 mM HEPES, pH 7.6 , 26 mM DTT, 7 mM NaF, 5 mM Mg(OAc)₂, 3 mM ATP, 0.05 mM FeCl₃, and either 10 μg of recombinant Pf R2 prepared by the method of Mann et al . Biochem. 30 1939-1947, or an equivalent volume of buffer A. For CDP reductase assays, 0.05 mM [5-³H]CDP (20 Ci/mol) is added to assay mix, while for GDP reductase assays 1.5 mM dTTP and 0.02 mM [2,8-³H]GDP (45Ci/mol) is added to a final volume of 100 μl. Reactions are initiated by the addition of recombinant Rl protein, and the assay mixture is incubated at 37°C for 5-15 min for initial rate determinations. Reactions are quenched by immersion in a boiling water bath for 4 min. Samples are frozen and lyophilized to dryness. Lyophilized samples are reconstituted in 1 ml of 50 mM Tris, pH 8.45 containing 100 mM Mg(OAc)₂ (buffer B) . Samples are centrifuged at 10,000 x G for 10 minutes to precipitate denatured protein. The supernatant is then loaded onto 0.5 x 5 cm columns of aminophenylboronate (Amicon) which had been pre-equilibrated with 5 ml of buffer B. Deoxynucleoside diphosphates elute in 5 ml (for dCDP) to 12 ml (for dGDP) of buffer B. Unreacted ribonucleoside diphosphate substrates can be quantitatively recovered and columns can be regenerated by treatment with 10 ml of 50 mM sodium citrate, pH 5.9. Radioactivity in aliquots of both buffer B and citrate fractions are determined by liquid scintillation counting.

B. Recombinant Pf R2 Activity

Pf R2 is reactivated by iron and tyrosyl radial regeneration using the procedures described by Mann, et al.. Biochem. 30 1939-1947 (1991)) . After reactivation, ribo- nucleotide reductase/Pf R2 activity is determined from the rate of reduction of [³H]-CDP. One unit is defined as the amount of protein which, in the presence of an excess of Rl subunit, catalyzes the formation of one nmol of dCDP/minutes at 37°C (Engstrδm et al . , Biochem. , 18 2941-2S48 (1979) ; Ingemarson et al .. J. Virol. 63 3769-3776 (1989)) . The reactivated Pf R2 protein is assayed in the presence of 15 μg of pure Pf Rl protein, obtained as described in Example 56, above.

Example 59

Detecting a Malarial Parasite in a Patient

The presence of a malarial parasite in a patient's blood is detected by a diagnostic polymerase chain reaction (PCR) assay via the following procedures. Red blood cells suspected of being infected with parasites are collected by centrifugation and lysed to release any malarial parasites that may be present as in Example 56. The DNA is extracted as in Example 56. A diagnostic PCR assay is then performed on the extracted DNA. Primers complementary to two regions of para¬ sitic DNA corresponding to a portion of SEQ ID NO:l shows divergence from the hosts' Rl and R2 are used for the - diagnostic PCR. PCR is carried out on Pf DNA in a standard PCR buffer with 1 μM of the upstream primer, 5' -ATT TTC CAT TCC AAA 3' (SEQ ID NO:41), and 1 μM of the downstream primer, 5'-AAA TTC GCT ATT CAG ACA-3' (SEQ ID NO:42) . The primers amplify a fragment of DNA about 196 nucleotides in length. Cycling is typically performed for 20-30 cycles at 94°C for 15 seconds, 50°C for 15 seconds, and 72°C for 15 seconds.

Analysis of the PCR products is performed on approximately 10% of the sample by running the sample portion on an 8.0% polyacrylamide. Gels are photographed and transferred to a solid support, e.g., ZetaProbe™ (BioRad) , following the manufacturer's recommended procedures to obtain a blot.

As a positive control, a blood sample which is infected by the malarial parasite is subjected to the same procedures as.described above. As a negative control, a blood sample which is free of any malarial parasites is subjected to the same procedures as described above.

Radioisotope labeled probes complementary to the portion of the Pf Rl or R2 DNA which the PCR primers will amplify are designed to probe the blots for the par-asitic DNA. The blot is exposed to the labelled probe under standard hydridization conditions. Presence of a blot bound to the probe indicates presence of the parasitic DNA.

All references cited with respect to synthetic, preparative and analytical procedures are incorporated herein by reference.

The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof and, accordingly, reference shouldbe made to the appended claims, rather than to the foregoing specification, as indicating the scope of the invention.

SEQUENCE LISTING (1) GENERAL INFORMATION:

(i) APPLICANT: The University of Pennsylvania (ii) INVENTORS: Barry S. Cooperman, Harvey Rubin, Jerome Salem and Alison L. Fisher

(iii) TITLE OF INVENTION: Plasmodium falciparum Ribonucleotide Reductase, DNA Sequences Therefor and Peptide Inhibitors Thereof

(iv) NUMBER OF SEQUENCES: 67 (v) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Seidel, Gonda, Lavorgna

& Monaco, P.C.

(B) STREET: Two Penn Center Plaza

Suite 1800

((CC)) CCIITTYY:: Philadelphia

(D) STATE: Pennsylvania

(E) COUNTRY: U.S.A.

(F) ZIP: 19103

(vi) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Diskette, 3.50 inch, 720 Kb

(B) COMPUTER: IBM PS/2

(C) OPERATING SYSTEM: MS-DOS

(D) SOFTWARE: WordPerfect 5.1 (vii) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: Not Yet Assigned

(B) FILING DATE: Not Yet Assigned

(C) CLASSIFICATION:

(viii) PRIORITY APPLICATION DATA:

(A) U.S. Application Serial No. 08/136,743 (B) U.S. Filing Date: 14 October 1993

(ix) ATTORNEY/AGENT INFORMATION:

(A) NAME: Monaco, Daniel A.

(B) REGISTRATION NUMBER: 30,480

(C) REFERENCE/DOCKET NUMBER: 3957-10 PC (x) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (215) 568-8383

(B) TELEFAX: (215) 568-5549

(C) TELEX: None (2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1112 nucleotides

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:

TCTAGAATCC AATATTTAGC AAACAGGAAA GAGAATTTAG TGATTTACAA 50

AAAGGGAAAG AGATAAATGA GAAGAATTTT AAATAAAGAG AGTGATCGAT 100 TTACTTTATA TCCAATATTA TATCCTGATG TTTTCCCATT TTACAAGAAA 150

GCTGAAGCTA GTTTTTGGAC AGCAGAAGAA ATTGATTATT CTAGTGATTT 200

AAAAGATTTT GAGAAATTGA ATGAAAATGA GAAACATTTT ATAAAGCATG 250

TGTTAGCATT TTTTGCAGCA AGTGATGGTA TAGTCTTAGA GAATTTGGCA 300

GTAAGTTTTT TAAGAGAGGT TCAAATAACA GAAGCTAAAA AATTTTATTC 350 CTTTCAAATA GCTGTAGAAA ATATTCATTC AGAAACATAT AGTTTATTAA 400

TTGATAATTA TATTAAAGAT GAAAAAGAAA GATTAAATTT ATTTCATGCT 450

ATAGAAAATA TCCCTGCCGT AAAAAATAAA GCATTATGGG CAGCAAAATG 500

GATTAACGAT ACTAATTCGT TTGCTGAAAG AATTGTTGCT AATGCATGTG 550

TTGAAGGAAT ATTATTCAGT GGTAGTTTTT GTGCAATTTT TTGGTTTAAG 600 AAACAAAATA AATTACACGG TTTGACATTT AGTAATGAAT TAATAAGTAG 650

AGATGAAGGA CTACATACAG ATTTTAATTG CTTAATTTAT AGTTTATTAG 700

ATAATAAACT TC^'CAGAACAA ATGGTACAAA ATATTGTTAA AGAAGCGGGG 750

GGTGTAGAAG TTGAAAAGTC TTTTATATGT GAATCCTTAC CATGTGATTT 800

AATAGGTATG AATTCTAGAC TTATGTCTCA ATATATAGAA TTTGTTGCTG 850 ATAGATTATT AGAATGCTTA GGATGCTCTA AAATTTTCCA TTCCAAAAAT 900

CCATTTAATT GGATGGACTT AATTTCACTT CAAGGAAAAA CAAACTTTTT 950

TGAGAAAAGA GTCGCAGATT ATCAAAAATC AGGAGTCATG GCTCAACGAA 1000

AGGATCATGT CTTTTGTCTG AATACGGAAT TTTAAATGAT ACTCGAAATA 1050

TTTATTATAC CATATGTATA CTATATAAAT ATATATATTA AATAATGATA 1100 GTATTTTTTT TT 1112

(3) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 322 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Arg Arg lie Leu Asn Lys Glu Ser Asp Arg Phe Thr Leu Tyr

5 10 15 Pro lie Leu Tyr Pro Asp Val Phe Pro Phe Tyr Lys Lys Ala Glu

20 25 30

Ala Ser Phe Trp Thr Ala Glu Glu lie Asp Tyr Ser Ser Asp Leu

35 40 45 Lys Asp Phe Glu Lys Leu Asn Glu Asn Glu Lys His Phe He Lys

50 55 60

His Val Leu Ala Phe Phe Ala Ala Ser Asp Gly He Val Leu Glu

65 70 75 Asn Leu Ala Val Ser Phe Leu Arg Glu Val Gin He Thr Glu Ala

80 85 90

Lys Lys Phe Tyr Ser Phe Gin He Ala Val Glu Asn He His Ser

95 100 105

Glu Thr Tyr Ser Leu Leu He Asp Asn Tyr He Lys Asp Glu Lys 110 115 120

Glu Arg Leu Asn Leu Phe His Ala He Glu Asn He Pro Ala Val

125 130 135

Lys Asn Lys Ala Leu Trp Ala Ala Lys Trp He Asn Asp Thr Asn

140 145 150 Ser Phe Ala Glu Arg He Val Ala Asn Ala Cys Val Glu Gly He

155 160 165

Lys Phe Ser Gly Ser Phe Cys Ala He Phe Trp Phe Lys Lys Gin

170 175 180

Asn Lys Leu His Gly Leu Thr Phe Ser Asn Glu Leu He Ser Arg 185 190 195

Asp Glu Gly Leu His Thr Asp Phe Asn Cys Leu He Tyr Ser Leu

200 205 210

Leu Asp Asn Lys Leu Pro Glu Gin Met Val Gin Asn He Val Lys

215 220 225 Glu Ala Gly Gly Val Glu Val Glu Lys Ser Phe He Cys Glu Ser

230 235 240

Leu Pro Cys Asp Leu He Gly Met Asn Ser Arg Leu Met Ser Gin

245 250 255

Thr He Glu Phe Val Ala Asp Arg Leu Leu Glu Cys Leu Gly Cys 260 265 270

Ser Lys He Phe His Ser Lys Asn Pro Phe Asn Trp Met Asp Lys

275 280 285

He Ser Leu Gin Gly Lys Thr Asn Phe Phe Glu Lys Arg Val Ala

290 295 300 Asp Tyr Gin Lys Ser Gly Val Met Ala Gin Arg Lys Asp His Val

305 310 315

Phe Cys Leu Asn Thr Glu Phe 320

(4) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2663 nucleotides (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GAGTACATCA GGAGATTAAG TGATGATGGT ATAAGGACCC CATCAGGTAA 50 ACCTATACAA ACAATGTATG TGTTGAATAG AAAGGGAGAA GAAGAAGATA 100 TATCCTTTGA TCAAATTTTA AAAAGAATAC AAAGGTTATC ATATGGTCTT 150 CATAAATTAG GTGAGTATCC TGCTTGTGTT ACACAAGGTG TAATCAATGG 200 GATGTATAGT AGTATAAAAA CCTGTGAATT AGATGAATTA GCTGCTCAAA 250 CATGTGCATA TATGGCTACA ACACATCCTG ATTTTTCTAT ATTAGCTGCA 300 CGTATTACTA CAGATAATTT GCACAAAAAT ACTAGTGATG ATGTTGCAGA 350 AGTAGCTGAA GCATTGTATA CGTATAAAGA TGGTAGAGGT AGACCAGCTA 400 GCTTAATTAG TAAGGAAGTA TATGATTTTA TTTTATTACA TAAAGTACGT 450 TTAAATAAAG AAATAGATTA TACTACCCAT TTTAATTATG ATTATTTTGG 500 ATTTAAAACA TTGGAAAGAT CTTATTTATT ACGTATTAAT AATAAAATTA 550 TTGAAAGACC TCAACATTTA TTAATGAGAG TTTCTATTGG TATACATATA 600 GATGACATAG ATAAAGCTTT AGAAACATAT CATTTAATGT CTCAGAAATA 650 TTTTACCCAT GCAACTCCTA CATTGTTTAA TTCAGGAACC CCAAGGCCAC 700 AAATGTCTTC TTGTTTCTTG TTATCAATGA AAGCAGATTC TATTGAAGGT 750 ATTTTTGAAA CTCTAAAACA ATGTGCTTTA ATTAGTAAAA CTGCAGGAGG 800 TATTGGTGTA GCAGTACAAG ATATAAGAGG ACAAAATTCT TATATTAGAG 850 GTACCAATGG AATATCTAAT GGTTTAGTAC CTATGTTAAG AGTTTTTAAT 900 GATACTGCAA GATATGTAGA TCAAGGTGGA GGAAAACGTA AGGGATCGTA 950 CGCTGTTTAT ATTGAACCAT GGCATTCAGA TATATTTGAA TTTTTAGATT 1000 TAAGAAAGAA TCATGGAAAA GAAGAATTAA GAGCACGAGA TTTATTTTAT 1050 GCTGTATGGG TTCCTGATCT TTTTATGAAG AGAGTTAAAG AAAATAAAAA 1100 TTGGACATTA ATGTGTCCAA ATGAATGTCC AGGTTTAAGT GAAACCTGGG 1150 GTGAAGAATT TGAAAAATTA TATACAAAAT ATGAAGAAGA AAATATGGGA 1200 AAAAAAACTG TGCTTGCTCA AGATTTATGG TTTGCTATAT TACAAAGCCA 1250 AATAGAAACA GGAGTACCCA TATATCTATA TAAAGATTCT TGTAATGCAA 1300

AACCAATCAA AAATTTAGGT ACAATTAAAT GTAGTAACTT ATGTTGTGAA 1350 ATAATCGAAT ATACCTCTCC TGATGAAGTT GCTGTATGTA ATTTGGCATC 1400 TATAGCTTTA TGTAAATTTG TAGATTTGGA AAAAAAAGAA TTCAATTTCA 1450 AAAAGTTATA TGAAATAACC AAAATTATTA CAAGAAATTT AGATAAAATT 1500 ATAGAAAGAA ATTATTATCC AGTCAAAGAA GCAAAAACAT CTAATACTAG 1550 ACATAGACCT ATTGGTATTG GTGTTCAAGG ATTAGCAGAC ACATTTATGT 1600 TATTAAGATA TCTTTATGAA TCTGATGCTG CAAAAGAATT GAATAAAAGA 1650 ATATATGAAA CTATGTATTA TGCTGCTTTA GAAATGTCGG TTGATTGGCT 1700 TCAATCTGGT CCATATGAAT CTTATCAAGG AAGTCCAGGT AGCCAAGGTA 1750 TATTACAATT TGATATGTGG AATGCTAAAG TTGATAACAA ATATTGGGAT 1800

TGGGATGAAT TAAAGCTAAA GATTGCAAAA ACTGGTTTAA GAAACCTATT 1850 ATTATTAGCA CCTATGCCAA CTGCATCTAC TTCACAAATT CTTGGAAACA 1900 ATGAATCCTT TGAACCATAT ACTAGTAATA TTTATTATAG AAGAGTTTTA 1950 AGTGGAGAAT TTTTCGTTGT TAATCCTCAT TTGTTAAAAG ATTTATTTGA 2000 CAGAGGTTTA TGGGATGAAG ACATGAAACA GCAATTAATA GCTCACAATG 2050

GAAGTATTCA ATATATAAGT GAAATACCAG ATGACTTGAA AGAGTTGTAC 2100 AAAACTGTAT GGGAAATTAA GCAAAAGAAT ATTATTGATA TGGCTGCAGA 2150 CAGGGGGTAT TTTATTGATC AGGTAAAATT AAAATATAAA AGATAATATA 2200 TATAAATA A AATAAATAAA TAAATAAATA AAAAAAAAAA TATATATATA 2250 TATATATATA TATATATTTA TATTTATAGT GTATGTCATT TGTTTTATAA 2300

GTATATATTT TGTTCATACA TTTATATTCA TATATATTTT TTTTCCTTTT 2350 TTTATAGTCC CAATCATTAA ATATTTATAT TCAAAAACCA ACCTTTGCAA 2400 AATTGTCAAG TATGCATTTC TATGGATGGG AAAAAGGATT GAAAACGGGA 2450 GCTTACTATT TAAGAACCCA AGCAGCGACC GATGCTATTA AATTTACCGT 2500 CGATACTCAT GTTGCAAAAA ATGCTGTAAA ACTCAAAAAT GCAGATGGAG 2550 TACAAATAAC AAGAGAAGTT TCCAGAGAAA CAATTCAACT GAATCAACGT 2600 TACTCAAAAT GTGTGTCCTT TAAGAGTAAT AATGATGAAC AATGTTTAAT 2650 GTGTTCTGGT TAA 2663

(5) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 811 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met Tyr Val Leu Asn Arg Lys Gly Glu Glu Glu Asp He Ser Phe

5 10 15

Asp Gin He Leu Lys Arg He Gin Arg Leu Ser Tyr Gly Leu His

20 25 30

Lys Leu Gly Glu Tyr Pro Ala Cys Val Thr Gin Gly Val He Asn 35 40 45

Gly Met Tyr Ser Ser He Lys Thr Cys Glu Leu Asp Glu Leu Ala

50 55 60

Ala Gin Thr Cys Ala Tyr Met Ala Thr Thr His Pro Asp Phe Ser 65 70 75 He Leu Ala Ala Arg He Thr Thr Asp Asn Leu His Lys Asn Thr

80 85 90

Ser Asp Asp Val Ala Glu Val Ala Glu Ala Leu Tyr Thr Tyr Lys

95 100 105

Asp Gly Arg Gly Arg Pro Ala Ser Leu He Ser Lys Glu Val Tyr 110 115 120

Asp Phe He Leu Leu His Lys Val Arg Leu Asn Lys Glu He Asp

125 130 135

Tyr Thr Thr His Phe Asn Tyr Asp Tyr Phe Gly Phe Lys Thr Leu

140 145 150 Glu Arg Ser Tyr Leu Leu Arg He Asn Asn Lys He He Glu Arg

155 160 165

Pro Gin His Leu Leu Met Arg Val Ser He Gly He His He Asp

170 175 180

Asp He Asp Lys Ala Leu Glu Thr Tyr His Leu Met Ser Gin Lys 185 190 195

Tyr Phe Thr His Ala Thr Pro Thr Leu Phe Asn Ser Gly Thr Pro

200 205 210

Arg Pro Gin Met Ser Ser Cys Phe Leu Leu Ser Met Lys Ala Asp

215 220 225 Ser He Glu Gly He Phe Glu Thr Leu Lys Gin Cys Ala Leu He

230 235 240

Ser Lys Thr Ala Gly Gly He Gly Val Ala Val Gin Asp He Arg

245 250 255

Gly Gin Asn Ser Tyr He Arg Gly Thr Asn Gly He Ser Asn Gly 260 265 270

Leu Val Pro Met Leu Arg Val Phe Asn Asp Thr Ala Arg Tyr Val

275 280 285

Asp Gin Gly Gly Gly Lys Arg Lys Gly Ser Tyr Ala Val Tyr He

290 295 300 Glu Pro Trp His Ser Asp He Phe Glu Phe Leu Asp Leu Arg Lys

305 310 315

Asn His Gly Lys Glu Glu Leu Arg Ala Arg Asp Leu Phe Tyr Ala

320 325 330 Val Trp Val Pro Asp Leu Phe Met Lys Arg Val Lys Glu Asn Lys

335 340 345

Asn Trp Thr Leu Met Cys Pro Asn Glu Cys Pro Gly Leu Ser Glu

350 355 360

Thr Trp Gly Glu Glu Phe Glu Lys Leu Tyr Thr Lys Tyr Glu Glu 365 370 375

Glu Asn Met Gly Lys Lys Thr Val Leu Ala Gin Asp Leu Trp Phe

380 385 390

Ala He Leu Gin Ser Gin He Glu Thr Gly Val Pro He Tyr Leu

395 400 405 Tyr Lys Asp Ser Cys Asn Ala Lys Pro He Lys Asn Leu Gly Thr

410 415 420

He Lys Cys Ser Asn Leu Cys Cys Glu He He Glu Tyr Thr Ser

425 430 435

Pro Asp Glu Val Ala Val Cys Asn Leu Ala Ser He Ala Leu Cys 440 445 450

Lys Phe Val Asp Leu Glu Lys Lys Glu Phe Asn Phe Lys Lys Leu

455 460 465

Tyr Glu He Thr Lys He He Thr Arg Asn Leu Asp Lys He He

470 475 480 Glu Arg Asn Tyr Tyr Pro Val Lys Glu Ala Lys Thr Ser Asn Thr

485 490 495

Arg His Arg Pro He Gly He Gly Val Gin Gly Leu Ala Asp Thr

500 505 510

Phe Met Leu Leu Arg Tyr Leu Tyr Glu Ser Asp Ala Ala Lys Glu 515 520 525

Leu Asn Lys Arg He Tyr Glu Thr Met Tyr Tyr Ala Ala Leu Glu

530 535 540

Met Ser Val Asp Trp Leu Gin Ser Gly Pro Tyr Glu Ser Tyr Gin

545 550 555 Gly Ser Pro Gly Ser Gin Gly He Leu Gin Phe Asp Met Trp Asn

560 565 570

Ala Lys Val Asp Asn Lys Tyr Trp Asp Trp Asp Glu Leu Lys Leu

575 580 585

Lys He Ala Lys Thr Gly Leu Arg Asn Leu Leu Leu Leu Ala Pro 590 595 600

Met Pro Thr Ala Ser Thr Ser Gin He Leu Gly Asn Asn Glu Ser

605 610 615

Phe Glu Pro Tyr Thr Ser Asn He Tyr Tyr Arg Arg Val Leu Ser

620 625 630 Gly Glu Phe Phe Val Val Asn Pro His Leu Leu Lys Asp Leu Phe

635 640 645

Asp Arg Gly Leu Trp Asp Glu Asp Met Lys Gin Gin Leu He Ala

650 655 660

His Asn Gly Ser He Gin Tyr He Ser Glu He Pro Asp Asp Leu 665 670 675

Lys Glu Leu Tyr Lys Thr Val Trp Glu He Lys Gin Lys Asn He

680 685 690

He Asp Met Ala Ala Asp Arg Gly Tyr Phe He Asp Gin Val Lys

695 700 705 Leu Lys Tyr Lys Arg Ser Gin Ser Leu Asn He Tyr He Gin Lys

710 715 720

Pro Thr Phe Ala Lys Leu Ser Ser Met His Phe Tyr Gly Trp Glu

725 730 735

Lys Gly Leu Lys Thr Gly Ala Tyr Tyr Leu Arg Thr Gin Ala Ala 740 745 750 Thr Asp Ala He Lys Phe Thr Val Asp Thr His Val Ala Lys Asn

755 760 765

Ala Val Lys Leu Lys Asn Ala Asp Gly Val Gin He Thr Arg Glu

770 775 780

Val Ser Arg Glu Thr He Gin Leu Asn Gin Arg Tyr Ser Lys Cys

785 790 795

Val Ser Phe Lys Ser Asn Asn Asp Glu Gin Cys Leu Met Cys Ser

800 805 810 Gly 811

(6) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Tyr Ala Gly Ala Val Val Asn Asp Leu 5

(7) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Phe Thr Leu Asp Ala Asp Phe

5

(8) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

Phe Cys Leu Asn Thr Glu Phe

5

(9) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

Gin Lys Ser Gly Val Met Ala Gin Arg Lys Asp His Val Phe Cys

5 10 15

Leu Asn Thr Glu Phe 20

(10) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

Glu Val Asp Thr Asp Asp Leu Ser Asn Phe Gin Leu 5 10

(11) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

Gly Gly Asn Thr Gly Asp Ser His Ala Xaa Phe Thr Leu Asp Ala 5 10 15

Asp Phe

(12) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

Lys Ser Thr Lys Gin Glu Ala Gly Ala Xaa Phe Thr Phe Asn Glu

5 10 15

Asp Phe

(13) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

Asn Ser Thr Glu Asn Ser Xaa Xaa Xaa Xaa Phe Thr Leu Asp Ala

5 10 15

Asp Phe

(14) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

Xaa Xaa Thr Glu Asn Ser Xaa Xaa Xaa Xaa Phe Thr Leu Asp Ala

5 10 15

Asp Phe

(15) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

Gin Glu Asp Asn His Xaa Xaa Xaa Xaa Xaa Phe Ser Leu Asp Val

5 10 15 sp Phe

(16) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

Thr Ser Tyr Ala Gly Ala Val Val Asn Asp Leu 5 10

(17) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

Thr Ser Tyr Ala Gly Thr Val He Asn Asp Leu 5 10 (18) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: GCWGAAGCWA GTTTTTGGAC A 21

(19) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 nucleotides

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: ACMCGTTTT TCAAACTTTG TTTT CC 27

(20) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: ATGCTAACAC ATGCTTT 17

(21) INFORMATION FOR SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: CTTTGTTTTT CCTTGAAGTG 20

(22) INFORMATION FOR SEQ ID NO:21: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 nucleotides

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

CTTATGTCTC AATATATAGA ATTTG 25

(23) INFORMATION FOR SEQ ID NO:22: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 nucleotides (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

GCATCCGAAG AATTTGAAMR YTRTAY 27

(24) INFORMATION FOR SEQ ID NO:23: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: ARWCCTTGWA GWCCWATWCC 20

(25) INFORMATION FOR SEQ ID NO:24: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: TATCCAGTCA AAGAAGCA 18

(26) INFORMATION FOR SEQ ID NO:25: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

CCATAAATCT TGAGCAAG 18

(27) INFORMATION FOR SEQ ID NO:26: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: Phe Thr Leu Asn Ala Asp Phe

5

(28) INFORMATION FOR SEQ ID NO:27: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: Phe Cys Leu Asp Ala Asp Phe

5

(29) INFORMATION FOR SEQ ID NO:28: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: Phe Cys Leu Asn Ala Asp Phe

5

(30) INFORMATION FOR SEQ ID NO:29: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: Phe Thr Leu Asp Thr Asp Phe

5

(31) INFORMATION FOR SEQ ID NO:30: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30

Phe Thr Leu Asn Thr Asp Phe 5

(32) INFORMATION FOR SEQ ID NO:31: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:

Phe Cys Leu Asp Thr Asp Phe 5

(33) INFORMATION FOR SEQ ID NO:32: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:

Phe Cys Leu Asn Thr Asp Phe 5

(34) INFORMATION FOR SEQ ID NO:33: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33

Phe Thr Leu Asp Ala Glu Phe 5

(35) INFORMATION FOR SEQ ID NO:34: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:

Phe Thr Leu Asn Ala Glu Phe 5

(36) INFORMATION FOR SEQ ID NO:35; (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: Phe Cys Leu Asp Ala Glu Phe

5

(37) INFORMATION FOR SEQ ID NO:36: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: Phe Cys Leu Asn Ala Glu Phe

5

(38) INFORMATION FOR SEQ ID NO:37: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: Phe Thr Leu Asp Thr Glu Phe

5

(39) INFORMATION FOR SEQ ID NO:38: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: Phe Thr Leu Asn Thr Glu Phe

5

(40) INFORMATION FOR SEQ ID NO:39: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: Phe Cys Leu Asp Thr Glu Phe

5 (41) INFORMATION FOR SEQ ID NO:40: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2433 nucleotides

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:

ATGTATGTGT TGAATAGAAA GGGAGAAGAA GAAGATATAT CCTTTGATCA 50

AATTTTAAAA AGAATACAAA GGTTATCATA TGGTCTTCAT AAATTAGGTG 100 AGTATCCTGC TTGTGTTACA CAAGGTGTAA TCAATGGGAT GTATAGTAGT 150

ATAAAAACCT GTGAATTAGA TGAATTAGCT GCTCAAACAT GTGCATATAT 200

GGCTACAACA CATCCTGATT TTTCTATATT AGCTGCACGT ATTACTACAG 250

ATAATTTGCA CAAAAATACT AGTGATGATG TTGCAGAAGT AGCTGAAGCA 300

TTGTATACGT ATAAAGATGG TAGAGGTAGA CCAGCTAGCT TAATTAGTAA 350 GGAAGTATAT GATTTTATTT TATTACATAA AGTACGTTTA AATAAAGAAA 400

TAGATTATAC TACCCATTTT AATTATGATT ATTTTGGATT TAAAACATTG 450

GAAAGATCTT ATTTATTACG TATTAATAAT AAAATTATTG AAAGACCTCA 500

ACATTTATTA ATGAGAGTTT CTATTGGTAT ACATATAGAT GACATAGATA 550

AAGCTTTAGA AACATATCAT TTAATGTCTC AGAAATATTT TACCCATGCA 600 ACTCCTACAT TGTTTAATTC AGGAACCCCA AGGCCACAAA TGTCTTCTTG 650

TTTCTTGTTA TCAATGAAAG CAGATTCTAT TGAAGGTATT TTTGAAACTC 700 TAAAACAATG TGCTTTAATT AGTAAAACTG CAGGAGGTAT TGGTGTAGCA 750 GTACAAGATA TAAGAGGACA AAATTCTTAT ATTAGAGGTA CCAATGGAAT 800 ATCTAATGGT TTAGTACCTA TGTTAAGAGT TTTTAATGAT ACTGCAAGAT 850 ATGTAGATCA AGGTGGAGGA AAACGTAAGG GATCGTACGC TGTTTATATT 900 GAACCATGGC ATTCAGATAT ATTTGAATTT TTAGATTTAA GAAAGAATCA 950 TGGAAAAGAA GAATTAAGAG CACGAGATTT ATTTTATGCT GTATGGGTTC 1000 CTGATCTTTT TATGAAGAGA GTTAAAGAAA ATAAAAATTG GACATTAATG 1050 TGTCCAAATG AATGTCCAGG TTTAAGTGAA ACCTGGGGTG AAGAATTTGA 1100 AAAATTATAT ACAAAATATG AAGAAGAAAA TATGGGAAAA AAAACTGTGC 1150

TTGCTCAAGA TTTATGGTTT GCTATATTAC AAAGCCAAAT AGAAACAGGA 1200 GTACCCATAT ATCTATATAA AGATTCTTGT AATGCAAAAC CAATCAAAAA 1250 TTTAGGTACA ATTAAATGTA GTAACTTATG TTGTGAAATA ATCGAATATA 1300 CCTCTCCTGA TGAAGTTGCT GTATGTAATT TGGCATCTAT AGCTTTATGT 1350 AAATTTGTAG ATTTGGAAAA AAAAGAATTC AATTTCAAAA AGTTATATGA 1400 AATAACCAAA ATTATTACAA GAAATTTAGA TAAAATTATA GAAAGAAATT 1450 ATTATCCAGT CAAAGAAGCA AAAACATCTA ATACTAGACA TAGACCTATT 1500 GGTATTGGTG TTCAAGGATT AGCAGACACA TTTATGTTAT TAAGATATCT 1550 TTATGAATCT GATGCTGCAA AAGAATTGAA TAAAAGAATA TATGAAACTA 1600 TGTATTATGC TGCTTTAGAA ATGTCGGTTG ATTGGCTTCA ATCTGGTCCA 1650 TATGAATCTT ATCAAGGAAG TCCAGGTAGC CAAGGTATAT TACAATTTGA 1700 TATGTGGAAT GCTAAAGTTG ATAACAAATA TTGGGATTGG GATGAATTAA 1750 AGCTAAAGAT TGCAAAAACT GGTTTAAGAA ACCTATTATT ATTAGCACCT 1800 ATGCCAACTG CATCTACTTC ACAAATTCTT GGAAACAATG AATCCTTTGA 1850 ACCATATACT AGTAATATTT ATTATAGAAG AGTTTTAAGT GGAGAATTTT 1900 TCGTTGTTAA TCCTCATTTG TTAAAAGATT TATTTGACAG AGGTTTATGG 1950 GATGAAGACA TGAAACAGCA ATTAATAGCT CACAATGGAA GTATTCAATA 2000 TATAAGTGAA ATACCAGATG ACTTGAAAGA GTTGTACAAA ACTGTATGGG 2050 AAATTAAGCA AAAGAATATT ATTGATATGG CTGCAGACAG GGGGTATTTT 2100 ATTGATCAGG TAAAATTAAA ATATAAAAGA TCCCAATCAT TAAATATTTA 2150 TATTCAAAAA CCAACCTTTG CAAAATTGTC AAGTATGCAT TTCTATGGAT 2200 GGGAAAAAGG ATTGAAAACG GGAGCTTACT ATTTAAGAAC CCAAGCAGCG 2250 ACCGATGCTA TTAAATTTAC CGTCGATACT CATGTTGCAA AAAATGCTGT 2300 AAAACTCAAA AATGCAGATG GAGTACAAAT AACAAGAGAA GTTTCCAGAG 2350 AAACAATTCA ACTGAATCAA CGTTACTCAA AATGTGTGTC CTTTAAGAGT 2400 AATAATGATG AACAATGTTT AATGTGTTCT GGT 2433

(42) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 nucleotides (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: ATT TTC CAT TCC AAA 15

(43) INFORMATION FOR SEQ ID NO:42: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 nucleotides

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: AAA TTC CGT ATT CAG ACA 18

(44) INFORMATION FOR SEQ ID NO:43: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43

Asn Ser Phe Thr Leu Asp Ala Asp Phe 5

(45) INFORMATION FOR SEQ ID NO:44: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:

Ser Phe Thr Leu Asp Ala Asp Phe 5 (46) INFORMATION FOR SEQ ID, NO:45: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:

Thr Leu Asp Ala Asp Phe 5

(47) INFORMATION FOR SEQ ID NO:46: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:

Leu Asp Ala Asp Phe 5

(48) INFORMATION FOR SEQ ID NO:47: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:

Leu Thr Leu Asp Ala Asp Phe 5

(49) INFORMATION FOR SEQ ID NO:48: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8

Phe Ser Leu Asp Ala Asp Phe 5

(50) INFORMATION FOR SEQ ID NO:49: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:

Phe Ala Leu Asp Ala Asp Phe 5

(51) INFORMATION FOR SEQ ID NO:50: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:

Phe Thr Val Asp Ala Asp Phe 5

(52) INFORMATION FOR SEQ ID NO:51: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51;

Phe Thr Phe Asp Ala Asp Phe 5

(53) INFORMATION FOR SEQ ID NO:52: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:

Phe Thr Ala Asp Ala Asp Phe 5 (54) INFORMATION FOR SEQ ID NO:53: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:

Phe Thr Leu Ala Ala Asp Phe 5

(55) INFORMATION FOR SEQ ID NO:54:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:

Phe Thr Leu Asp Gly Asp Phe 5

(56) INFORMATION FOR SEQ ID NO:55:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:

Phe Thr Leu Asp Leu Asp Phe 5

(57) INFORMATION FOR SEQ ID NO:56:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:

Phe Thr Leu Asp Ala Glu Phe 5

(58) INFORMATION FOR SEQ ID NO:57:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:

Phe Thr Leu Asp Ala Asn Phe 5

(59) INFORMATION FOR SEQ ID NO:58: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:

Phe Thr Leu Asp Ala Lys Phe 5

(60) INFORMATION FOR SEQ ID NO:59:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:

Phe Thr Leu Asp Ala Ala Phe 5

(61) INFORMATION FOR SEQ ID NO:60:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:

Phe Thr Leu Asp Ala Asp Leu 5

(62) INFORMATION FOR SEQ ID NO:61:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:

Phe Leu Asp Ala Asp Phe 5

(63) INFORMATION FOR SEQ ID NO:62: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:62

Phe Thr Leu Asp Asp Phe 5

(64) INFORMATION FOR SEQ ID NO:63: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:

Phe Thr Leu Asp Phe 5

(65) INFORMATION FOR SEQ ID NO:64: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:

Phe Thr Leu Asp Ala Asp Phe Ala Ala 5

(66) INFORMATION FOR SEQ ID NO:65: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:

Ala Gly Ala Phe Thr Phe Asn Glu Asp Phe 5

(67) INFORMATION FOR SEQ ID NO:66: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:

Phe Thr Phe Asn Glu Asp Phe 5 (68) INFORMATION FOR SEQ ID NO:67: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:67;

Thr Phe Asn Glu Asp Phe 5

Claims (34)

1. A purified DNA segment encoding for Pf Rl or Pf

   R2,
2. 2. A DNA segment consisting essentially of a nucleotide sequence encoding for a polypeptide according to SEQ ID NO: 2 or SEQ ID NO:4.
3. 3. A DNA segment according to claim 2 wherein said DNA segment comprises a nucleotide sequence according to SEQ ID NO:l, SEQ ID NO:3, or SEQ ID NO:40.
4. 4. A DNA seqment according to claim 2 wherein said

   DNA segment comprises a nucleotide sequence according to SEQ ID NO:40.
5. 5. A purified Pf Rl or Pf R2 polypeptide.
6. 6. A polypeptide having essentially an amino acid sequence according to SEQ ID NO:2 or SEQ ID NO:4.
7. 7. A plasmid transfer or storage vector comprising a DNA segment according to claim 2.
8. 8. A host cell transformed by a vector comprising a DNA segment according to claim 2, which host cell under culture conditions is capable of producing a polypeptide according to SEQ ID NO:2 or SEQ ID NO:4.
9. 9. A method for producing a polypeptide having an amino acid sequence essentially according to SEQ ID NO:2 or SEQ ID NO:4, comprising: cloning into a host cell a DNA segment encoding for a polypeptide having essentially the amino acid sequence according to SEQ ID NO:2 or SEQ ID NO:4, culturing said host cell under such conditions so as to produce said polypeptide, and isolating or purifying said polypeptide from the culture media and/or said host cells.
10. 10. A compound having a chain length from seven to about 400 amino acids and having the formula:

    Y-X₁-Phe-X₃-Leu-X₄-Phe-X₂;

    wherein Y is H or a blocking group on the peptide N-terminal amino group; X_x is from zero to about 393 amino acids, provid¬ ed when X_x is more than one amino acid, the amino acids are either the same or different; X₂ is OH or at least one amino acid, provided when X₂ is more than one amino acid, the amino acids are either the same of different; X₃ is any amino acid; X₄ is from zero to three amino acids, provided when X₄ is more than one amino acid, the amino acids are either the same or different; and further providing that the total amino acids of the sequence corresponding to the segment -Leu-X₄-Phe-X₂ of formula (I) is five amino acids; or a pharmaceutically acceptable salt thereof.
11. 11. A compound according to claim 10, wherein X₁ is from zero to about 93 amino acids.
12. 12. A compound according to claim 10, wherein X₁ is from zero to about 13 amino acids.
13. 13. A compound according to claim 10, wherein the blocking group is an acyl group.
14. 14. A compound according to claim 13, wherein X_x is from zero to about three amino acids and the acyl group is an acetyl or benzoyl group.
15. 15. A compound according to claim 10, wherein X₄ is a member selected from the group consisting of one of the following sequences:

    Xaa-Xaa-Xaa;

    Xaa-Xaa-Glu; Xaa-Thr-Xaa Asn-Xaa-Xaa Xaa-Thr-Glu Asn-Xaa-Glu Asn-Thr-Xaa and

    Asn-Thr-Glu; wherein each Xaa of the X₄ group is the same or different and is independently any amino acid.
16. 16. A compound according to claim 15, wherein X₃ is

    Cys.
17. 17. A compound according to claim 15, comprising an amino acid sequence selected from the group consisting of:

    SEQ ID NO:7, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,

    SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34,

    SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39.
18. 18. An antimalarial peptide according to claim 17 comprising an amino acid sequence according to SEQ ID NO:7.
19. 19. An antimalarial composition comprising a pharmaceutically acceptable carrier and at least one compound according to claim 10.
20. 20. An antimalarial composition comprising a pharma¬ ceutically acceptable carrier and at least one compound according to claim 17.
21. 21. A method for controlling the proliferation of Plasmodium falciparum comprising: administering to a patient an effective amount of at least one compound having a chain length from seven to about 400 amino acids and having the formula:

    Y-X₁-Phe-X₃-Leu-X₄-Phe-X₂; wherein Y is H or a blocking group on the peptide N-terminal amino group; X_x is from zero to about 393 amino acids, provid¬ ed when X-_L is more than one amino acid, the amino acids are either the same or different; X₂ is OH or at least one amino acid, provided when X₂ is more than one amino acid, the amino acids are either the same of different; X₃ is any amino acid; X₄ is from zero to three amino acids, provided when X₄ is more than one amino acid, the amino acids are either the same or different; and further providing that the total amino acids of the sequence corresponding to the segment -Leu-X₄-Phe-X₂ of formula (I) is five amino acids; or a pharmaceutically acceptable salt thereof.
22. 22. A method according to claim 21, wherein X₄ is a member selected from the group consisting of one of the following sequences : Xaa-Xaa-Xaa; Xaa-Xaa-Glu Xaa-Thr-Xaa

    Asn-Xaa-Xaa Xaa-Thr-Glu Asn-Xaa-Glu Asn-Thr-Xaa and

    Asn-Thr-Glu,^• wherein each Xaa of the X₄ group is the same or different and is independently any amino acid.
23. 23. A method according to claim 22, wherein X₃ is Cys.
24. 24. A method according to claim 22, wherein said compound comprises an amino acid sequence selected from the group consisting of:

    SEQ ID NO:7,

    SEQ ID NO:26,

    SEQ ID NO:27, SEQ ID NO:28,

    SEQ ID NO:29,

    SEQ ID NO:30,

    SEQ ID NO:31,

    SEQ ID NO:32, SEQ ID NO:33,

    SEQ ID NO:34,

    SEQ ID NO:35,

    SEQ ID NO:36,

    SEQ ID NO:37, SEQ ID NO:38, and

    SEQ ID NO:39.
25. 25. A method according to claim 24, wherein said compound comprises an amino acid sequence according to SEQ ID NO:7.
26. 26. A compound according to claim 10, wherein Y is H; X_x is from zero to about three amino acids; and the total chain length of the compound is from seven to about ten amino acids.
27. 27. An antimalarial composition according to 19, which composition inhibits Pf RR reduction of ribonucleo-tides to 2'deoxyribonucleotides at an inhibition rate of as least twice the rate at which it inhibits human RR reduction of ribonucleotides to 2'deoxyribonucleotides .
28. 28. A recombinant Pf RR enzyme comprising recombinant Pf Rl, having a sequence according to SEQ ID NO:4, or a functionally equivalent polypeptide thereof; and recombinant Pf R2, having a sequence according to SEQ ID NO:2, or a functional equivalent thereof.
29. 29. A recombinant Pf RR enzyme according to claim 28, wherein said recombinant Pf RR enzyme is of the formula (Pf R2)₂-(Pf Rl)₂ and said Pf R2 and Pf Rl of said formula comprise a sequence according to SEQ ID NO:2 and SEQ ID NO:4, respectively.
30. 30. A method of screening a potential anti-malarial agent for antimalarial activity which comprises contacting said potential antimalarial agent with a recombinant Pf RR enzyme according to claim 29 and assaying for inhibition of Pf RR enzymatic activity.
31. 31. A method of screening a potential anti-malarial agent for antimalarial activity which comprises contacting said potential antimalarial agent with recom-binant Pf Rl and assaying for binding of said agent to Pf Rl, wherein said binding step is optionally carried out in the presence of recombinant Pf R2.
32. 32. A method according to claim 31, comprising contacting said potential antimalarial agent with recombinant

    Pf Rl in the presence of recombinant Pf R2, and assaying for the agents ability to block the binding of Pf Rl and Pf R2.
33. 33. A method according to claim 31, comprising contacting said potential antimalarial agent with recom-binant

    Pf Rl, adding recombinant Pf R2, and assaying for the ability of the agent to block the binding of Pf Rl and Pf R2.
34. 34. A method of diagnosing the presence or absence of a malarial parasitic infection in a host which comprises : a) obtaining a blood sample from a host in need of diagnosis for the presence or absence of a malarial parasite; b) contacting said sample with a PCR primer pair which is capable of amplifying a target Rl or R2 DNA segment of said malarial parasite, which target DNA segment is unique as compared to said host DNA, under conditions such that said primer pair will amplify said target DNA segment if said target DNA segment is present; c) contacting said sample with a polynucleotide probe which is capable of specifically hybridizing with said target DNA segment, under conditions such that said poly- nucleotide probe will hybridize with said target DNA segment if said target DNA segment is present; and d) assaying said sample to determine the presence or absence of said malarial parasite.